Polypeptides binding to human complement C5

ABSTRACT

The present invention relates to C5 binding polypeptides, comprising a C5 binding motif, BM, which motif consists of an amino acid sequence selected from i) EX 2 X 3 X 4 A X 6 X 7 EID X 11 LPNL X 16 X 17 X 18 QW X 21 AFIX 25 X 26 LX 28 D, and ii) an amino acid sequence which has at least 86% identity to the sequence defined in i), wherein the polypeptide binds to C5. The present invention moreover relates to C5 binding polypeptides for use in therapy, such as for use in treatment of a C5 related condition, and to methods of treatments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a U.S. National Stage Application ofPCT/SE2013/050139 filed Feb. 19, 2013 which is incorporated by referencein its entirety.

TECHNICAL FIELD

The present disclosure relates to polypeptides that bind to humancomplement component 5 (C5) and to the use of such polypeptides intherapy.

BACKGROUND

The complement protein C5 is a central component of the complementsystem; a key part of the innate immune system. The complement system isan intricate immune survival system with numerous tasks in tightlycontrolled, diverse processes. One of its functions is as first linehost defense against infection by other organisms by discriminatinghealthy host tissues from cellular debris and apoptotic and necroticcells. Furthermore, it is involved in clearance of immune complexes,regulation of the adaptive immune response, promotion of tissueregeneration, angiogenesis, mobilization of stem cells and developmentof the central nervous system (Woodruff et al. Mol Immunol 2011, 48(14):1631-1642); Ricklin et al. Nat Immunol 2010, 11(9):785-795). Anytrigger, for example erroneous or unrestricted activation orinsufficient regulation, that disturbs the fine balance of complementactivation and regulation may lead to pathologic conditions includingself-attack of the host's cells leading to extensive tissue damage.

The complement system consists of about 30 proteins. There are threepathways to initiate complement immunity; the classical pathway thatemploys C1q to recognize immune complexes on the surface of cells, thelectin pathway that is initiated when mannose-binding lectin (MBL)recognizes certain sugars and the alternative pathway that is initiatedspontaneously by hydrolysis of complement factor 3 (C3), a processsuppressed by certain mammalian cell surface molecules not present oninvading pathogens. The alternative pathway also acts as anamplification loop for the complement system. All three pathwaysconverge at the level of C3. Cleavage of C3 into C3a and C3b leads tothe formation of a convertase that in turn cleaves complement factor 5(C5) into C5a and C5b. C5a is a very potent attractant of various immunecells while C5b oligomerizes with C6-9 to form a pore known as themembrane attack complex (MAC) or sometimes the terminal complementcomplex (TCC). Activation of the complement system leads to a number ofmechanisms with the purpose of neutralizing the pathogen; formation ofMAC on the surface of a cell such as an invading bacteria lead to lysis,deposition of C3 and C4 cleavage products C3b and C4b aids opsonizationleading to phagocytosis of the pathogen by macrophages andanaphylatoxins such as C3a and C5a attracts monocytes and neutrophils tothe site of activation, upregulates surface markers leading to increasedimmunologic susceptibility and to the release of cytokines.

C5 is a 190-kDa glycoprotein comprised of 2 disulfide-linked polypeptidechains, alpha and beta, with a molecular mass of 115 and 75 kDa,respectively (Tack et al. Biochem 1979, 18:1490-1497). Haviland et al.(J Immun 1991, 146: 362-368) constructed the complete cDNA sequence ofhuman complement pro-05, which is predicted to encode a 1,676-amino acidpro-molecule that contains an 18-amino acid leader peptide and a 4-aminoacid linker separating the beta and alpha chains. Blockade of C5cleavage into C5a and C5b prevents MAC formation and formation of thepro-inflammatory C5a but leaves the upstream complement effector systemintact allowing the C3/C4 mediated opsonization.

The complement system's key role in the defense against pathogens ingeneral makes it an interesting target for pharmaceutical intervention.This is emphasized by the fact that many mutations or impairedregulation of complement is involved in various diseases and conditions.These include increased susceptibility to auto-immune diseases such assystemic lupus erythematosis (SLE) where deposition of immune complexestriggers the classical pathway (Manderson et al. Annu Rev Immunol 2004,22:431-456). In addition, mutations of the complement proteins C1-C5often result in SLE or SLE like symptoms. Other autoimmune diseases witha strong involvement of the complement system are rheumatoid arthritis(RA) where immune complexes may activate complement in the RA joint,Sjögren's syndrome, dermatomyositis and other autoantibody drivendiseases such as Guillain-Barré syndrome (GBS), Fisher syndrome (Kaidaet al. J. Neuroimmun 2010, 223:5-12) different types of vasculitis,systemic sclerosis, anti-glomerular basement membrane (anti-GBM) andanti-phospholipid syndrome (APS) (Chen et al. J Autoimmun 2010,34:J276-J286).

The complement system is furthermore involved in neurodegenerativedisorders such as Alzheimer's disease (AD) where Aβ plaques directlyactivate the complement system leading to C5a mediated recruitment ofmicroglia. This was further confirmed when a C5aR antagonist was shownto be neuroprotective in a mouse model of AD (Fonseca et al. J Immunol2009, 183:1375-1383). Auto-antibodies against the acetylcholine receptorand subsequent complement activation is the most common cause tomyasthenia gravis, a disease that affects the neuromuscular junction(Toyka and Gold, Schweizer Archive Neurol Psych 2007, 158:309-321). MACformation is involved in the pathophysiology of multiple sclerosis (MS)(Oh et al. Immunol Res 2008, 40:224-234). Also in Parkinson's disease,Huntington's disease and prion diseases such as Creutzfeld-Jacobdisease, complement activation is a part of the pathology (Bonifati andKishore, Mol Immunol 2007, 44:999-1010). In wound healing, inflammatoryresponses are a key component to restore tissue homeostasis and thecomplement system is involved in the early recognition of damagedtissue. However, in models of chronic wounds and severe burns, forexample, inhibition of complement by e. g. C1 inhibitor resulted inimproved healing and decreased tissue damage suggesting that complement.Furthermore, various complement deficiencies, such as exemplified by theC4 knockout mouse, have been found to be protective against long-termtissue damage resulting from wounds (reviewed in Cazender et al.Clinical and Developmental Immunology 2012, on-line publication). Latelyit has been shown that tumor growth and proliferation is facilitated bycomplement activation, in particular by C5a, and that blockade of theC5a receptor slows down this process. In addition, mice lacking C3display significantly slower tumor growth than wild-type littermates(Markiewski et al. Nat Immunol 2008, 9:1225-1235).

Dysfunctional complement regulation is the cause of several rare toultra-rare conditions, such as paroxysmal nocturnal hemoglobinuria (PNH)and atypical hemolytic uremic syndrome (aHUS), where hemolysis is a keyfeature in the pathology. In PNH, a clone of hematopoetic stem cellswith mutated PIG-A gene encoding phosphatidylinositolN-acetylglucosaminyltransferase subunit A take over the pool of bloodcells. This mutation leads to loss of GPI anchored proteins such as thecomplement regulators CD55 and CD59. Red blood cells lacking CD55 andCD59 on the surface are exposed for complement mediated lysis by MAC.Clinically, PNH is manifested by hemolysis leading to anemia, thrombosisand bone marrow failure. Atypical HUS is caused by mutations inregulatory proteins of mainly the alternative pathway, such as bymutations in factor H.

The eye is strongly indicated as a site for complement driven pathology.The most common cause of visual loss is age-related macular degeneration(AMD) where, in its more severe form (exudative or wet AMD), pathologicchoridal neurovascular membranes develop under the retina. In the US,about 10% of the population aged 65-74 shows sign of maculardegeneration and as many as 5% have visual impairment as a result toAMD. These numbers increase dramatically with age, but there are alsogenetic factors. Among the genes strongest associated with AMD arecomplement factor H, factor B and C3 and the C1 inhibitor (Bradley etal. Eye 2011, 25:683-693). Furthermore, several studies and clinicaltrials using various complement blocking molecules have provenbeneficial, suggesting that a C5 blocking molecule could help thesepatient groups. However, the current treatments of advanced AMD aims atinhibition of vascular endothelial growth factor (VEGF) inducedvascularization by intravitreal injections of e.g. Ranibizumab (amonoclonal antibody fragment) and Bevacizumab (monoclonal antibody). Inanimal models of uveitis, inflammation of the eye due to immuneresponses to ocular antigens, blocking antibodies against alternativepathway factor B (Manickam et al. J Biol Chem 2011, 286:8472-8480) aswell as against C5 (Copland et al. Clin Exp Immunol 2009, 159:303-314),improved the disease state.

In transplantation of solid organs, there are two major mechanisticpathways leading to rejection or delayed/impaired function of thegraft: 1) the immunologic barriers between donor and recipient withrespect to blood group (ABO) and MHC classes as well as extent ofpre-sensitization of the recipient against the donor, i.e. occurrence ofdonor specific antibodies (DSA) leading to acute antibody mediatedrejection (AMR); and 2) the condition of the transplanted organ as wellas the period of time it has been kept without constant blood perfusion,i.e. the degree of ischemic damage or ischemia reperfusion injury (IRI)of the graft. In both AMR and IRI, the complement system is attackingthe organ recognized as foreign and, therefore, an entity that should berejected. In AMR, the pre-existing anti-donor antibodies rapidly formimmune complexes on the surface of the foreign organ leading torecognition by C1q and subsequent activation of the complement systemvia the classical pathway. This process, known as hyper-acute rejectionhappens within minutes and, therefore modern transplantation ofmismatched organs includes elimination of DSA prior to transplantationby plasmapheresis or plasma exchange and intravenous IgG combined withdifferent immunosuppressants. Novel treatments also include B-celldepletion via usage of the anti-CD20 antibody Rituximab (Genberg et al.Transplant 2008, 85:1745-1754). These protocols have vastly eliminatedthe occurrence of hyper-acute rejection but still, in highly sensitizedpatients, the incidence of acute AMR (weeks-months) is as high as 40%(Burns et al. Am J Transplant 2008, 6:2684-2694; Stegall et al. Am JTransplant 2011, early on-line publication). With respect to IRI, mostevidence points at the terminal pathway with subsequent MAC formationand lysis as the main cause of tissue damage. Thus, a C5 blockingpolypeptide would be protective against rejection regardless of thecause being AMR, IRI or, as often happens, a combination of both AMR andIRI. As expected, highly perfused organs, such as the liver (Qin et al.Cell Mol Immunol 2006, 3:333-340), the heart and the kidneys areparticularly susceptible to complement mediated damage.

The central placement of the C5 protein; connecting the proximal and theterminal parts of the complement cascade, makes it an attractive targetfor pharmaceutical intervention. Since C5 is common to all pathways ofcomplement activation, blocking of C5 will stop the progression of thecascade regardless of the stimuli and thereby prevent the deleteriousproperties of terminal complement activation while leaving theimmunoprotective and immunoregulatory functions of the proximalcomplement cascade intact.

Antibodies targeted to human complement C5 are known from, e.g., WO95/29697; WO 02/30985; and WO 2004/007553. Eculizumab (SOLIRIS) is ahumanized monoclonal antibody directed against protein C5 and preventscleavage of C5 into C5a and C5b. Eculizumab has been shown to beeffective in treating PNH, a rare and sometimes life threatening diseaseof the blood characterized by intravascular hemolytic anemia,thrombophilia and bone marrow failure, and is approved for thisindication. Eculizumab was also recently approved by the FDA fortreatment of atypical hemolytic syndrome (aHUS), a rare but lifethreatening disease caused by loss of control of the alternativecomplement pathway leading to over-activation manifested as thromboticmicroangiopathy (TMA) leading to constant risk of damage to vital organssuch as kidney, heart and the brain. In aHUS, transplantation of thedamaged organ only temporarily helps the patient as the liver continuesto produce the mutated form of controlling protein (most oftencomplement factor H or other proteins of the alternative pathway). Arelated disease with a transient acute pathophysiology is HUS caused byinfection of Shiga toxin positive E. coli (STEC-HUS) and there arepromising clinical data suggesting efficacy also for this condition(Lapeyraque et al, N Engl J Med 2011, 364:2561-2563). Finally, the C5blocking antibody Eculizumab has proven efficacious in preventing AMR inrecipients of highly mismatched kidneys (Stegall, M. D. et al. Am JTransplant 2011, 11:2405-2413).

Apart from full length antibodies, single-chain variable fragments(scFV), minibodies and aptamers targeting C5 are described inliterature. These C5 inhibitors may bind to different sites (epitopes)on the C5 molecule and may have different modes of action. For example,whereas Eculizumab interacts with C5 at some distance of the convertasecleavage site, the minibody MUBODINA interacts with the cleavage site ofC5. The C5 inhibitory protein Ornithodoros moubata Complement Inhibitor(OmCI, Nunn, M. A. et al. J Immunol 2005, 174:2084-2091) from soft ticOrnithodoros moubata has been hypothesized to bind to the distal end ofthe CUB-C5d-MG8 superdomain, which is close to the convertase cleavagesite (Fredslund et al. Nat Immunol 2008, 9 (7):753-760). In contrast tothe three proteins mentioned above inhibiting cleavage of C5, themonoclonal antibody TNX-558 binds to a C5a epitope present both onintact C5 and released C5a without inhibiting the cleavage of C5. (Funget al. Clin Exp Immunol 2003, 133 (2):160-169).

Antibodies with their large, multidomain structure, 12 intra-chain and 4inter-chain disulfide bridges and complex glycosylation patterns, have anumber of intrinsic disadvantages related to their molecular structure.For example, the size of Eculizumab is about 148 kDa. The concentrationof C5 in human blood is about 400 nM and in order to block C5 activityentirely, the concentration of the inhibitor must be at least equal orhigher than that. Therefore, the standard life-long treatment regimen ofPNH using SOLIRIS is intravenous infusions of 900 mg protein everysecond week, a treatment that mainly take place in the clinic leading togreat inconvenience to the patient and cost to the society. SOLIRIS hasalso been reported to cause chest pain, fever, chills, itching, hives,flushing of the face, rash, dizziness, troubled breathing, or swellingof the face, tongue, and throat, although the reasons for these sideeffects are not clear. Furthermore, Eculizumab is not active in anytested animal model, including primates, making animal studies with theactive drug impossible. As mentioned above, the current treatments ofAMD are also antibody dependent and, thus, treatments based oninjections or other routes of administration with molecules of lowermolecular weight, are highly required.

In addition, antibody production is more difficult and more expensivethan production of small proteins (Kenanova et al. Expert Opin DrugDeliv 2006, 3 (1):53-70). Other drawbacks generally related toantibodies are listed by Reilly et al. (Clin Pharmacokinet 1995,28:126-142), such as cross-reactivity and non-specific binding to normaltissues, increased metabolism of injected antibodies and formation ofhuman anti-human antibodies (HAMA) causing decreased or loss of thetherapeutic effect.

Thus, continued provision of agents with comparable C5 blocking activityremains a matter of substantial interest within the field. Inparticular, there is a continued need for molecules that prevent theterminal complement cascade as well as the formation of thepro-inflammatory molecule C5a. Of great interest is also a provision ofuses of such molecules in the treatment of disease.

DESCRIPTION

It is an object of the invention to provide new C5 binding agents. It ismoreover an object of the invention to provide new C5 binding agents foruse in therapeutic applications.

In one aspect, there is provided a C5 binding polypeptide, comprising aC5 binding motif, BM, which motif consists of the amino acid sequenceselected from

i) (SEQ ID NO. 763)EX₂X₃X₄A X₆X₇EID X₁₁LPNL X₁₆X₁₇X₁₈QW X₂₁AFIX₂₅ X₂₆LX₂₈D,

wherein, independently of each other,

X₂ is selected from H, Q, S, T and V;

X₃ is selected from I, L, M and V;

X₄ is selected from A, D, E, H, K, L, N, Q, R, S, T and Y;

X₆ is selected from N and W;

X₇ is selected from A, D, E, H, N, Q, R, S and T;

X₁₁ is selected from A, E, G, H, K, L, Q, R, S, T and Y;

X₁₆ is selected from N and T;

X₁₇ is selected from I, L and V;

X₁₈ is selected from A, D, E, H, K, N, Q, R, S and T;

X₂₁ is selected from I, L and V;

X₂₅ is selected from D, E, G, H, N, S and T;

X₂₆ is selected from K and S;

X₂₈ is selected from A, D, E, H, N, Q, S, T and Y;

and

ii) an amino acid sequence which has at least 86% identity to thesequence defined in i), wherein the polypeptide binds to C5.

The above defined class of sequence related polypeptides having abinding affinity for C5 is derived from a common parent polypeptidesequence. More specifically, the definition of the class is based on ananalysis of a large number of random polypeptide variants of the parentpolypeptide that were selected for their interaction with C5 inselection experiments. The identified C5 binding motif, or “BM”,corresponds to the target binding region of the parent scaffold, whichregion constitutes two alpha helices within a three-helical bundleprotein domain. In the parent scaffold, the varied amino acid residuesof the two BM helices constitute a binding surface for interaction withthe constant Fc part of antibodies. By random variation of bindingsurface residues and subsequent selection of variants, the Fcinteraction capacity of the binding surface has been replaced with acapacity for interaction with C5.

As accounted for in the following Examples, selection of C5 bindingpolypeptide variants may for example be achieved by phage display forselection of naïve variants of a protein scaffold optionally followed byaffinity maturation and cell display for selection of affinity maturatedC5 binding variants. It is however understood that any selection system,whether phage-based, bacterial-based, cell-based or other, may be usedfor selection of C5 binding polypeptides.

The terms “C5 binding” and “binding affinity for C5” as used in thisspecification refers to a property of a polypeptide which may be testedfor example by the use of surface plasmon resonance technology, such asin a Biacore instrument (GE Healthcare). C5 binding affinity may e.g. betested in an experiment in which C5 is immobilized on a sensor chip of aBiacore instrument, and the sample containing the polypeptide to betested is passed over the chip. Alternatively, the polypeptide to betested is immobilized on a sensor chip of the instrument, and a samplecontaining C5, or fragment thereof, is passed over the chip. The skilledperson may then interpret the results obtained by such experiments toestablish at least a qualitative measure of the binding of thepolypeptide to C5. If a quantitative measure is desired, for example todetermine the apparent equilibrium dissociation constant K_(D) for theinteraction, surface plasmon resonance methods may also be used. Bindingvalues may for example be defined in a Biacore 2000 instrument (GEHealthcare). C5 is immobilized on a sensor chip of the measurement, andsamples of the polypeptide whose affinity is to be determined areprepared by serial dilution and injected over the chip. K_(D) values maythen be calculated from the results using for example the 1:1 Langmuirbinding model of the BIAevaluation software provided by the instrumentmanufacturer. The C5 or fragment thereof used in the K_(D) determinationmay for example comprise the amino acid sequence represented by SEQ IDNO:760.

In one embodiment of the C5 binding polypeptide according to the presentinvention, the C5 binding polypeptide binds to C5 such that the K_(D)value of the interaction is at most 1×10⁻⁶ M, such as at most 1×10⁻⁷ M,1×10⁻⁸ M, or 1×10⁻⁹ M.

A C5 binding polypeptide according to the present invention may be usedas an alternative to conventional antibodies or low molecular weightsubstances in various medical, veterinary and diagnostic applications.In particular, the C5 binding polypeptide may be useful in any methodrequiring affinity for C5 of a reagent. Accordingly, the C5 bindingpolypeptide may be used as a detection reagent, a capture reagent, aseparation reagent, a diagnostic agent or a therapeutic agent in suchmethods.

As the skilled person will realize, the function of any polypeptide,such as the C5 binding capacity of the polypeptides as defined herein,is dependent on the tertiary structure of the polypeptide. It istherefore possible to make minor changes to the amino acid sequence of apolypeptide without largely affecting the tertiary structure and thefunction thereof. Thus, in one embodiment, the polypeptide comprisesmodified variants of the BM of i), which are such that the resultingsequence is at least 89% identical to a sequence belonging to the classdefined by i), such as at least 93% identical, such as at least 96%identical to a sequence belonging to the class defined by i). Forexample, it is possible that an amino acid residue belonging to acertain functional grouping of amino acid residues (e.g. hydrophobic,hydrophilic, polar etc) could be exchanged for another amino acidresidue from the same functional group.

In another embodiment of the C5 binding polypeptide as defined above,the amino acid sequence is selected from i) as defined above, and iii)an amino acid sequence which in the 13 variable positions as denoted byX_(n), wherein n is 2-4, 6-7, 11, 16-18, 21, 25-26 and 28, has at least84% identity to the sequence defined in i), and which in positions 1, 5,8-10, 12-15, 19-20, 22-24, 27 and 29 has at least 87% identity to thesequence defined in i).

In one embodiment of the polypeptide according to the present invention,X₂ is selected from H, T and V. In another embodiment, X₂ is selectedfrom T and V. In yet another embodiment, X₂ is V.

In one embodiment of the polypeptide according to the present invention,X₃ is selected from I, L and V. In another embodiment, X₃ is selectedfrom I and L. In yet another embodiment, X₃ is I. In an alternativeembodiment, X₃ is L.

In one embodiment of the polypeptide according to the present invention,X₄ is selected from A, D, E, K, L, Q and R. In another embodiment, X₄ isselected from A, D, E, K and R. In yet another related embodiment, X₄ isselected from D and E.

In one embodiment of the polypeptide according to the present invention,X₆ is W.

In one embodiment of the polypeptide according to the present invention,X₇ is selected from A, D, N and T. In another embodiment, X₇ is selectedfrom D and N. In yet another related embodiment, X₇ is D. In analternative embodiment, X₇ is N.

In one embodiment of the polypeptide according to the present invention,X₁₁ is selected from A, H, K, Q, R and S. In another embodiment, X₁₁ isselected from A, H, K and R. In yet another related embodiment, X₁₁ isselected from A, K and R. In yet another related embodiment, X₁₁ isselected from K and R.

In one embodiment of the polypeptide according to the present invention,X₁₆ is T.

In one embodiment of the polypeptide according to the present invention,X₁₇ is selected from I and L. In another embodiment, X₁₇ is I. In analternative embodiment, X₁₇ is L.

In one embodiment of the polypeptide according to the present invention,X₁₈ is selected from A, D, E, N, Q, S and T. In another embodiment, X₁₈is selected from A, D, E, Q and S. In yet another related embodiment,X₁₈ is selected from D, E and Q. In yet another related embodiment, X₁₈is selected from D and E. In yet another related embodiment, X₁₈ is D.In an alternative embodiment, X₁₈ is E.

In one embodiment of the polypeptide according to the present invention,X₂₁ is selected from I and L. In another embodiment, X₂₁ is I. In analternative embodiment, X₂₁ is L.

In one embodiment of the polypeptide according to the present invention,X₂₅ is selected from E, H, N and T. In another embodiment, X₂₅ isselected from E and N. In yet another related embodiment, X₂₅ is N.

In one embodiment of the polypeptide according to the present invention,X₂₆ is K.

In one embodiment of the polypeptide according to the present invention,X₂₈ is selected from A, D, E, H, N, Q and S. In another embodiment ofthe above disclosed polypeptide, X₂₈ is selected from A, D, E and S. Inyet another related embodiment, X₂₈ is selected from A, D and E. In yetanother related embodiment, X₂₈ is selected from D and E. In yet anotherrelated embodiment, X₂₈ is D.

In one embodiment of the polypeptide according to the present invention,X₃X₄ is selected from LE and LD.

In one embodiment of the polypeptide according to the present invention,X₁₇X₁₈ is selected from IE and LD.

In the above embodiments of the first aspect, examples of C5 bindingpolypeptides falling within the class of polypeptides are identified. Itis contemplated that the above individual embodiments may be combined inall conceivable ways and still fall within the scope of the presentinvention. Such combinations of individual embodiments define arestricted, in one or more of the positions X₂-X₂₈, amino acid sequenceas compared to the amino acid definition in i).

The above embodiments of a C5 binding polypeptide may for example becombined such that the amino acid i) fulfils at least four of thefollowing eight conditions I-VIII:

I. X₂ is V;

II. X₃ is selected from I and L;

III. X₆ is W;

IV. X₇ is selected from D and N;

V. X₁₇ is selected from I and L;

VI. X₂₁ is L;

VII. X₂₅ is N;

VIII. X₂₈ is D.

In some examples of a C5 binding polypeptide according to the firstaspect, the amino acid sequence i) fulfils at least five of the eightconditions I-VIII. More specifically, the amino acid sequence i) mayfulfill at least six of the eight conditions I-VIII, such at least sevenof the eight conditions I-VIII, such as all of the eight conditionsI-VIII.

As described in the following Examples, the selection of C5 bindingvariants has led to the identification of individual C5 binding motif(BM) sequences. These sequences constitute individual embodiments of C5binding polypeptides according to this aspect. The sequences ofindividual C5 binding motifs are presented in FIG. 1 and as SEQ IDNO:1-248. In some embodiments of this aspect, the BM sequence i) isselected from any one of SEQ ID NO:1-12, SEQ ID NO:20, SEQ ID NO:23-24,SEQ ID NO:26-28, SEQ ID NO:32-35, SEQ ID NO:38-39, SEQ ID NO:41, SEQ IDNO:46, SEQ ID NO:49, SEQ ID NO:56-57, SEQ ID NO:59, SEQ ID NO:66, SEQ IDNO:78-79, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:106, SEQ ID NO:110, SEQID NO:119, SEQ ID NO:125, SEQ ID NO:141, SEQ ID NO:151, SEQ ID NO:161,SEQ ID NO:166, SEQ ID NO:187, SEQ ID NO:197, SEQ ID NO:203, SEQ IDNO:205, SEQ ID NO:215 and SEQ ID NO:243. More specifically, the BMsequence i) is selected from any one of SEQ ID NO:1-12, such as from SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. Inparticular, the BM sequence i) may be selected from SEQ ID NO:1 and SEQID NO:4.

In particular embodiments, the C5 binding motif (BM) forms part of athree-helix bundle protein domain. For example, the BM may essentiallyconstitute two alpha helices with an interconnecting loop, within saidthree-helix bundle protein domain.

The three-helix bundle protein domain is, in another embodiment,selected from domains of bacterial receptor proteins. Non-limitingexamples of such domains are the five different three-helical domains ofProtein A from Staphylococcus aureus, such as domain B, and derivativesthereof. In some embodiments, the three-helical bundle protein domain isa variant of protein Z, which is derived from said domain B ofstaphylococcal Protein A.

In embodiments where the C5 binding polypeptide of the invention formspart of a three-helix bundle protein domain, the C5 binding polypeptidemay comprise an amino acid sequence selected from:

i) (SEQ ID NO. 764) K-[BM]-DPSQS X_(a)X_(b)LLX_(c) EAKKL NDX_(d)Q;wherein[BM] is a C5 binding motif as defined above;X_(a) is selected from A and S;X_(b) is selected from N and E;X_(c) is selected from A, S and C;X_(d) is selected from A and S;andii) an amino acid sequence which has at least 79% identity to any one ofthe sequences defined above. Said amino acid sequence may have at least81%, such as at least 83%, such as at least 85%, such as at least 87%,such as at least 89%, such as at least 91%, such as at least 93%, suchas at least 95%, such as at least 97% identity to any one of thesequences defined above.

In one embodiment of the C5 binding polypeptide as defined above, X_(a)is A. In an alternative embodiment of the C5 binding polypeptide asdefined above, X_(a) is S.

In one embodiment of the C5 binding polypeptide as defined above, X_(b)is N. In an alternative embodiment, X_(b) is E.

In one embodiment of the C5 binding polypeptide as defined above, X_(b)is A. In an alternative embodiment, X_(c) is S. In yet anotheralternative embodiment, X_(c) is C.

In one embodiment of the C5 binding polypeptide as defined above, X_(d)is A. In an alternative embodiment, X_(d) is S.

In one embodiment of the C5 binding polypeptide as defined above, X_(a)is A; X_(b) is N; X_(c) is A and X_(d) is A.

In a further embodiment of the C5 binding polypeptide as defined above,X_(a) is A; X_(b) is N; X_(c) is C and X_(d) is A.

In a further embodiment of the C5 binding polypeptide as defined above,X_(a) is S; X_(b) is E; X_(c) is S and X_(d) is S.

In a further embodiment of the C5 binding polypeptide as defined above,X_(a) is S; X_(b) is E; X_(c) is C and X_(d) is S.

In yet a further embodiment, the amino acid sequence of the C5 bindingpolypeptide as defined above is selected from SEQ ID NO:249-496, inparticular from SEQ ID NO:249-260, SEQ ID NO:268, SEQ ID NO:271-272, SEQID NO:274-276, SEQ ID NO:280-283, SEQ ID NO:286-287, SEQ ID NO:289, SEQID NO:294, SEQ ID NO:297, SEQ ID NO:304-305, SEQ ID NO:307, SEQ IDNO:314, SEQ ID NO:326-327, SEQ ID NO:335, SEQ ID NO:340, SEQ ID NO:354,SEQ ID NO:358, SEQ ID NO:367, SEQ ID NO:373, SEQ ID NO:389, SEQ IDNO:399, SEQ ID NO:409, SEQ ID NO:414, SEQ ID NO:435, SEQ ID NO:445, SEQID NO:451, SEQ ID NO:453, SEQ ID NO:463 and SEQ ID NO:491, such as fromSEQ ID NO:249-260. In a further embodiment, the amino acid sequence isselected from SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252and SEQ ID NO:253, such as from SEQ ID NO:249 and SEQ ID NO:252.

Thus, in a further embodiment, there is provided a C5 bindingpolypeptide which comprises an amino acid sequence selected from:

i) (SEQ ID NO 765) YAK-[BM]-DPSQS SELLX_(c) EAKKL NDSQA P;wherein [BM] is a C5 binding motif as defined above andX_(c) is selected from S and C; andii) an amino acid sequence which has at least 81% identity to any one ofthe sequences defined in i) above.

Alternatively, there is provided a C5 binding polypeptide whichcomprises an amino acid sequence selected from:

i) (SEQ ID NO. 766) FNK-[BM]-DPSQS ANLLX_(c) EAKKL NDAQA P;wherein [BM] is a C5 binding motif as defined above andX_(c) is selected from A and C; andii) an amino acid sequence which has at least 81% identity to any one ofthe sequences defined in i) above.

As discussed above, polypeptides comprising minor changes as compared tothe above amino acid sequences without largely affecting the tertiarystructure and the function thereof are also within the scope of thepresent application. Thus, in some embodiments, the C5 bindingpolypeptides as defined above may for example have a sequence which isat least 83%, at least 84%, at least 86%, at least 88%, at least 90%, atleast 92%, at least 94%, at least 96% or at least 98% identical to thesequence defined in i).

In some embodiments and as disclosed in the Examples below, the C5binding motif may form part of a 58 or 60 amino acid polypeptide. Such apolypeptide may e.g. comprise a sequence selected from any one of SEQ IDNO:497-757, in particular a sequence selected from any one of SEQ IDNO:497-508, SEQ ID NO:516, SEQ ID NO:519-520, SEQ ID NO:522-524, SEQ IDNO:528-531, SEQ ID NO:534-535, SEQ ID NO:537, SEQ ID NO:542, SEQ IDNO:545, SEQ ID NO:552-553, SEQ ID NO:555, SEQ ID NO:562, SEQ IDNO:574-575, SEQ ID NO:583, SEQ ID NO:588, SEQ ID NO:602, SEQ ID NO:606,SEQ ID NO:615, SEQ ID NO:621, SEQ ID NO:637, SEQ ID NO:647, SEQ IDNO:657, SEQ ID NO:662, SEQ ID NO:683, SEQ ID NO:693, SEQ ID NO:699, SEQID NO:701, SEQ ID NO:711, SEQ ID NO:739 and SEQ ID NO:745-757, such as asequence selected from SEQ ID NO:497-508 and SEQ ID NO:745-757. Inanother embodiment, the amino acid sequence is selected from SEQ IDNO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500, SEQ ID NO:501, SEQID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:750 and SEQ IDNO:753, such as from any one of SEQ ID NO:497, SEQ ID NO:500, SEQ IDNO:748 and SEQ ID NO:753.

Binding of a molecule to C5 does not necessarily inhibit cleavage of C5.Inhibition is dependent on binding site, and since it is not entirelyclear what effects interaction with specific regions of C5 have, some C5binding molecules may interact with C5 without inhibiting its cleavageinto C5a and C5b. In one embodiment of the present invention, the C5binding polypeptide, when e.g. administered to a mammalian subject,inhibits cleavage of C5. The C5 binding polypeptide according to theinvention may more specifically inhibit cleavage of human C5 whenadministered to a human subject.

The structure of C5 differs somewhat between species, and thus, a C5binder found to bind C5 of one species may be inactive in anotherspecies. The humanized antibody Eculizumab, for example, binds to adomain of C5 often referred to as MG7. This region is highly variablebetween species and, therefore, Eculizumab binding is restricted tohuman C5. The C5 binding polypeptide of the present invention, however,is not restricted to human C5 but exhibits activity in animal models aswell, as demonstrated in the appended Examples.

The skilled person will understand that various modifications and/oradditions can be made to a C5 binding polypeptide according to anyaspect disclosed herein in order to tailor the polypeptide to a specificapplication without departing from the scope of the present invention.For example, a C5 binding polypeptide according to any aspect maycomprise further C terminal and/or N terminal amino acids. Such apolypeptide should be comprehended as a polypeptide having additionalamino acids residues at the very first and/or the very last position inthe polypeptide chain, i.e. at the N- and/or C-terminus. Thus, a C5binding polypeptide may comprise any suitable number of additional aminoacid residues, for example at least one additional amino acid residue.Each additional amino acid residue may individually or collectively beadded in order to, for example, improve production, purification,stabilization in vivo or in vitro, coupling, or detection of thepolypeptide. Such additional amino acid residues may comprise one ormore amino acid residues added for the purpose of chemical coupling. Oneexample of this is the addition of a cysteine residue. Such additionalamino acid residues may also provide a “tag” for purification ordetection of the polypeptide, such as a His₆ tag or a “myc” (c-myc) tagor a “FLAG” tag for interaction with antibodies specific to the tag orimmobilized metal affinity chromatography (IMAC) in the case of theHis₆-tag.

The further amino acids as discussed above may be coupled to the C5binding polypeptide by means of chemical conjugation (using knownorganic chemistry methods) or by any other means, such as expression ofthe C5 binding polypeptide as a fusion protein.

The further amino acids as discussed above may for example comprise oneor more polypeptide domain(s) A further polypeptide domain may providethe C5 binding polypeptide with another function, such as for exampleanother binding function, or an enzymatic function, or a toxic function(e.g. an immunotoxin), or a fluorescent signaling function, orcombinations thereof.

A further polypeptide domain may moreover provide the C5 bindingpolypeptide with the same binding function. Thus, in a furtherembodiment, there is provided a C5 binding polypeptide comprising atleast two C5 binding polypeptide monomer units, the amino acid sequencesof which may be the same or different. Multimeric forms of thepolypeptides may comprise a suitable number of domains, each having a C5binding motif, and each forming a “monomer” within the multimer. Thesedomains may all have the same amino acid sequence, but alternatively,they may have different amino acid sequences. In particular, the C5binding polypeptide of the invention may form homo- or heterodimers.

The further polypeptide domain(s) as described above may be joined tothe C5 binding polypeptide by covalent coupling using known organicchemistry methods. Alternatively, the C5 binding polypeptide comprisingthe further polypeptide domain(s) may be expressed as one or more fusionpolypeptides, for example in a system for recombinant expression ofpolypeptides, or joined in any other fashion, either directly or via alinker, for example an amino acid linker.

In some embodiments, the further polypeptide domain(s) may comprise ahalf-life extending moiety which increases the half life of the C5binding polypeptide in vivo. As understood by the skilled person,increased, or extended, half life means slower clearance of a particularmolecule from blood. There are a number of known strategies forprolonging the half life of a particular polypeptide in vivo, such ascoupling to the Fc domain of an antibody (Fc conjugation) or coupling toalbumin Another example is coupling to a half life extending moiety,e.g. a peptide or protein, that will associate to serum albumin in vivo.In particular, the half life extending moiety may be an albumin bindingmoiety. An albumin binding moiety may e.g. consist of a naturallyoccurring polypeptide, or an albumin binding fragment thereof, or anengineered polypeptide. An engineered polypeptide may be derived from anaturally occurring starting polypeptide through subjecting it toprotein engineering techniques, such as mutations and alterations in asite-directed or randomized approach, with a view to create novel orenhanced properties, such as binding affinity for a molecule such asalbumin. Such an engineered albumin binding polypeptide may for examplebe a variant of a protein scaffold, which variant has been selected forits specific binding affinity for albumin. In a specific embodiment, theprotein scaffold may be selected from domains of streptococcal Protein Gor derivatives thereof, such as for example domain GA1, domain GA2 anddomain GM of Protein G from Streptococcus strain G148, in particulardomain GA3.

Accordingly, in one embodiment of the C5 binding polypeptide, thefurther amino acids improves stabilization in vivo or in vitro andcomprise an albumin binding domain (ABD) of streptococcal protein G, ora derivative thereof. One example of an albumin binding domain which maybe comprised as a further polypeptide domain in the C5 bindingpolypeptide of the invention is set out in SEQ ID NO:759. Other examplesof suitable albumin binding domains are disclosed in WO 2009/016043 andWO 2012/004384. Such an ABD-extended polypeptide binds to serum albuminin vivo, and benefits from its longer half life, which increases the nethalf life of the polypeptide itself (see e.g. WO 91/01743). Thepharmacokinetic profile of a C5 binding polypeptide comprising analbumin binding moiety as defined above thus resembles that of serumalbumin when administered for example to a mammalian subject. ABD andderivatives thereof bind very strongly to human serum albumin (HSA) aswell as to serum albumin from other species, such as mouse and rat.

ABD of streptococcal protein G is 46 amino acid long, and thus when a C5binding polypeptide according to the invention comprises an ABD moietyor a derivative thereof, the overall size of the C5 binding polypeptideis relatively small. When administered for example to a mammaliansubject, such as a human subject, the albumin binding part of the C5binding polypeptide will associate non-covalently with serum albumin andthe polypeptide may thereby benefit from decreased renal clearance andincreased recirculation in epithelial cells. Tissue penetration mayhowever still be fast due to extravasating properties of serum albumin.Furthermore, a C5 binding polypeptide comprising a half life extendingmoiety may not only display an extended half life in vivo, but also areduced immunologic response in vivo, as compared to a polypeptidelacking a corresponding half life extending moiety (see e.g. WO2005/097202).

In a related aspect, there is provided a C5 binding compound, comprisingat least one C5 binding polypeptide according to any preceding claim; atleast one albumin binding domain of streptococcal protein G, or aderivative thereof, and at least one linking moiety for linking said atleast one domain or derivative thereof to the C or N terminus of said atleast one C5 binding polypeptide. Such a C5 binding compound has highaffinity for C5 as well as for serum albumin in vivo, when administerede.g. to a mammalian subject, and binding to serum albumin does notinterfere with the interaction with C5, as demonstrated in the followingExamples.

In one embodiment, the C5 binding compound has a structure selected from

[CBP1]-[L1]-[ALBD];

[CBP1]-[CBP2]-[L1]-[ALBD];

[CBP1]-[L1]-[ALBD]-[L2]-[CBP2];

[ALBD]-[L1]-[CBP1];

[ALBD]-[L1]-[CBP1]-[CBP2];

[CBP1]-[L1]-[CBP2]-[L2]-[ALBD]; and

[ALBD]-[L1]-[CBP1]-[L2]-[CBP2]

wherein, independently of each other,

[CBP1] and [CBP2] are C5 binding polypeptides which may be the same ordifferent;

[L1] and [L2] are linking moieties which may be the same or different;and

[ALBD] is an albumin binding domain of streptococcal protein G, orderivative thereof.

Preferred C5 binding compounds have a structure selected from

[CBP1]-[CBP2]-[L1]-[ALBD];

[CBP1]-[L1]-[ALBD]-[L2]-[CBP2]; and most preferably,

[CBP1]-[L1]-[ALBD].

Examples of linking moieties that may be used in such C5 bindingcompounds are selected from G, GS; [G₂S]_(n); [G₃S]_(n) (SEQ ID NO:783); [G₄S]_(n) (SEQ ID NO: 784); GS[G₄S]_(n) (SEQ ID NO: 785), whereinn is 0-7 (preferably, n is 0-2); [S₂G]_(m); [S₃G]_(m) (SEQ ID NO: 786);[S₄G]_(m) (SEQ ID NO: 787); wherein m is 0-7, and VDGS (SEQ ID NO: 788).Preferred linkers are GS and GS[G₄S]₂ (SEQ ID NO: 785).

Examples of albumin binding domains or derivatives thereof that may becomprised in a C5 binding compound are as described above. Inparticular, one example of an albumin binding domain is set out in SEQID NO:759.

Particularly preferred C5 binding compounds have the structure[CBP1]-[L1]-[ALBD], wherein [CBP1] is a polypeptide selected from SEQ IDNO:748 and SEQ ID NO:753, [L1] is GS, and [ALBD] is a polypeptide shownas SEQ ID NO:759.

The C5 binding polypeptide(s) comprised in a C5 binding polypeptide are,in one embodiment, independently selected from 58-mer or 60-mer C5binding polypeptides as previously described. In particular, the C5binding compound may comprise one or more C5 binding polypeptidesindependently selected from any one of SEQ ID NO:497-508, SEQ ID NO:516,SEQ ID NO:519-520, SEQ ID NO:522-524, SEQ ID NO:528-531, SEQ IDNO:534-535, SEQ ID NO:537, SEQ ID NO:542, SEQ ID NO:545, SEQ IDNO:552-553, SEQ ID NO:555, SEQ ID NO:562, SEQ ID NO:574-575, SEQ IDNO:583, SEQ ID NO:588, SEQ ID NO:602, SEQ ID NO:606, SEQ ID NO:615, SEQID NO:621, SEQ ID NO:637, SEQ ID NO:647, SEQ ID NO:657, SEQ ID NO:662,SEQ ID NO:683, SEQ ID NO:693, SEQ ID NO:699, SEQ ID NO:701, SEQ IDNO:711, SEQ ID NO:739 and SEQ ID NO:746-757, such as a sequence selectedfrom SEQ ID NO:497-508 and SEQ ID NO:746-757. In another embodiment, theamino acid sequence is selected from SEQ ID NO:497, SEQ ID NO:498, SEQID NO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:746, SEQ ID NO:747,SEQ ID NO:748, SEQ ID NO:750 and SEQ ID NO:753, such as from any one ofSEQ ID NO:497, SEQ ID NO:500, SEQ ID NO:748 and SEQ ID NO:753.

In a further aspect, there is provided a polynucleotide encoding a C5binding polypeptide or a compound as described above. An expressionvector comprising such a polynucleotide may enable production of a C5binding polypeptide or a C5 binding compound, for example by expressionin a host cell.

It should be understood that the C5 binding polypeptide according to thepresent invention may be useful as a therapeutic or diagnostic agent inits own right or as a means for targeting other therapeutic ordiagnostic agents, with e.g. direct or indirect effects on thecomplement protein C5. A direct therapeutic effect may for example beaccomplished by inhibiting C5 cleavage. In one embodiment, there is thusprovided a combination of a C5 binding polypeptide or a C5 bindingcompound according to the invention with a therapeutic agent.Non-limiting examples of therapeutic agents that may prove useful insuch a combination are immunostimulatory agents and radionuclides.

Thus, the C5 binding polypeptide as such, or as comprised in a C5binding compound or a combination according to the invention, is in oneembodiment provided for use in therapy, for example for the treatment ofa C5 related condition, such as a C5 related condition in a mammal, suchas a human subject. In one embodiment, said C5 related condition isselected from inflammatory disease, such as antigen-induced arthritis,sepsis, synovial inflammation, vasculitis and asthma; autoimmunedisease, such as systemic lupus erythematosus (SLE), cold agglutinindisease, rheumatoid arthritis, multiple sclerosis (MS), Sjögren'ssyndrome, dermatomyositis, myasthenia gravis and other autoantibodydriven diseases such as Guillain-Barré syndrome (GBS), Fisher syndrome,systemic sclerosis, anti-glomerular basement membrane (anti-GBM) andanti-phospholipid syndrome (APS); infectious disease, such ashemolytic-uremic syndrome (HUS), viral infections, bacterial infectionsand fungal infections; cardiovascular disease, such as (acute)myocardial infarction (undergoing revascularization either byfibrinolysis or percutaneous coronary intervention (PCI));neurodegenerative disorders such as Alzheimer's disease (AD),Huntington's disease, Creutzfeld-Jacob disease and Parkinson's disease;cancers; wounds; graft injury, such as ischemia reperfusion injury (IRI)and acute antibody mediated rejection (AMR); eye disease, such asage-related macular degeneration (AMD), uveitis, diabetic oculardiseases and disorders, and retinopathy of prematurity; kidney disease,such as membranous glomerulonephritis, membranous nephritis,immunoglobulin A nephropathy, Lupus nephritis, Goodpasture syndrome andpost-streptococcal glomerulonephritis; pulmonary diseases, such as adultrespiratory distress syndrome, chronic obstructive pulmonary disease andcystic fibrosis; hematological diseases; such as hemolytic anaemia,paroxysmal cold hemoglobinuria, atypical hemolytic uremic syndrome(aHUS) and paroxysmal nocturnal hemoglobinuria (PNH); allergic diseases,such as anaphylactic shock, allergy and asthma; and dermatologicaldiseases, such as pemphigus, bullous pemphigoid, phototoxic reactionsand psoriasis. In a more particular embodiment, the C5 bindingpolypeptide, compound or combination according to the invention is usedfor treatment of paroxysmal nocturnal hemoglobinuria (PNH).

As mentioned when discussing organ transplantation in the backgroundsection above, differences between donor and recipient (e.g. ABO and MHCclasses) as well as the condition of the transplanted organ may lead todelayed functioning or even rejection of the transplanted organ.Treatment may thus be necessary to eliminate anti-donor antibodiesdespite a positive donor-recipient crossmatch or to eliminate ABOantibodies when transplantation occurs against the ABO barrier. Suchtreatment typically includes immunoadsorption, e.g. by use of affinitychromatography techniques, prior to as well as after transplantation orplasmapheresis. Such procedures however runs the risk of eliminatingnearly all antibodies present in the circulation, thus includingtherapeutic antibodies. The C5 binding polypeptides or compounds of theinvention are however not affected by any antibody removing procedures,and may thus be exploited in these treatments.

In some C5 related conditions where a more local acute pathology inreadily accessible tissues, such as lung and the blood stream, dominatesrather than systemic pathologies, a drug with a very short half-lifecould be advantageous over one with a slow elimination. Thus, in such C5related conditions, a C5 binding polypeptide without a half-lifeextending moiety may be beneficial. As previously accounted for, a C5binding polypeptide according to the invention will, due to itsrelatively small size, exhibit a relatively rapid pharmacokineticprofile when administered to a mammal such as a human. The C5 bindingpolypeptide according to the invention may potentially be active intreatment of C5 related conditions such as asthma (Zhang et al. ExpertRev Clin Immunol 2010, 6:269-277), sepsis (Ward et al. The Sci World J2010, 10:2395-2402), and hypersensitivity syndrome including the Cactivation-related pseudoallergy (CARPA, a reaction to certaintherapeutic liposomes and lipid excipient-based drugs that in rare casescan lead to life threatening cardiopulmonary distress (Szebeni et al.Adv Drug Delivery Rev 2011, 63:1020-1030). In addition, a C5 bindingpolypeptide according to the invention may be used for complementinhibition when a recipient of blood transfusion has received blood ofan incompatible type (a situation occurring in about 1:14000 transfusionunits in the US which is associated with high mortality, Goodnough etal. Lancet 2003, 361:161-169).

In a related aspect, there is provided a method of treatment of a C5related condition, comprising administering of a C5 binding polypeptide,or combination as described above to a mammalian subject in needthereof. Consequently, in the method of treatment, the subject istreated with a C5 binding polypeptide, a C5 binding compound or acombination according to the invention. In a more specific embodiment ofsaid method, the binding of the C5 binding polypeptide or thecombination, to a C5 expressed on a cell surface in the subject inhibitsC5 cleavage. In one embodiment of the method of treatment, the C5related condition is selected from inflammatory disease; autoimmunedisease; infectious disease; cardiovascular disease; neurodegenerativedisorders; cancers; wounds; graft injury; eye disease; kidney disease;pulmonary diseases; hematological diseases; allergic diseases anddermatological diseases. In particular the C5 related condition may beas defined above in relation to therapeutic use of a C5 bindingpolypeptide, compound or combination according to the invention. The C5related condition may for example be paroxysmal nocturnal hemoglobinuria(PNH). In one embodiment of the method of treatment, the said C5 bindingpolypeptide is administered intravenously, subcutaneously, byinhalation, nasally, orally, intravitreally, or topically.

The invention will now be further illustrated by the followingnon-limiting Examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1AH is a listing of the amino acid sequences of examples of C5binding motifs comprised in C5 binding polypeptides of the invention(SEQ ID NO:1-248), examples of 49-mer C5 binding polypeptides accordingto the invention (SEQ ID NO:249-496), examples of 58-mer C5 bindingpolypeptides according to the invention (SEQ ID NO:497-744) and examplesof 60-mer C5 binding polypeptides according to the invention (SEQ IDNO:745-757), as well as the sequences of protein Z (SEQ ID NO:758), analbumin binding domain (ABD094, SEQ ID NO:759), the Swiss-Prot entryP01031 of human C5 (amino acid residues 1-1676, SEQ ID NO:760; of whichthe α-chain corresponds to amino acid residues 678-1676 and the β-chaincorresponds to amino acid residues 19-673), the sequence of theHis₆-tagged tic protein OmCI used herein (SEQ ID NO:761) and cynomolgusC5 (SEQ ID NO:762) derived from genomic sequence (published on-line atwww.ebi.ac.uk/ena; Ebeling et al. (2001) Genome Res. 21(10):1746-1756)using human C5 as template. The sequence contains two unknown aminoacids “X” in positions 63 and 1346.

FIG. 2 shows the result of a typical binding analysis performed in aBiacore instrument as described in Example 2. Sensorgrams were obtainedby injection of human C5 (hC5; black solid curve), cynomolgus C5 (cC5;black short-dashed curve), rat C5 (rC5; black long-dashed curve), humanMG7 domain (hMG7; gray dotted curve), and human immunoglobulin G (hIgG;gray solid curve), respectively, over an immobilized dimeric Z variant(Z05477, SEQ ID NO:509).

FIG. 3 is a column chart showing the response in ELISA against hC5 andrC5, respectively, for selected maturated Z variants. The black columnscorresponds to the absorbance at 450 nm obtained using 0.05 μg/ml hC5(left column in each group) and to the absorbance at 450 nm obtainedusing 4 μg/ml rC5 for each Z variant (right column in each group), asdescribed in Example 4. The responses for the Z variant Z05363 (SEQ IDNO:510) are plotted as a positive control.

FIG. 4 schematically shows different constructs encompassing one orseveral C5 binding Z variants selected from SEQ ID NO:745-757,optionally linked to ABD094 (SEQ ID NO:759).

FIG. 5 shows SDS-PAGE analyses of purified C5 binding Z variants(reduced condition) visualized by Instant Blue, as described in Example6. A) represents one example of dimeric Z-Z-ABD (lane 1 where Z is equalto SEQ ID NO:745 and ABD is equal to SEQ ID NO:759) compared withdifferent Z-ABD fusion proteins (where Z is equal to SEQ ID NO:745 (lane2), SEQ ID NO:748-757 (lanes 4-13) fused to ABD094 (SEQ ID NO:759) by aGS linker); B) represents one C5 binding Z variant (SEQ ID NO:753) indifferent constructs, and C) represents two different C5 binding Zvariants (SEQ ID NO:748, lanes 2-3 and 6 and SEQ ID NO:753, lanes 4-5and 7), in monomeric form (lanes 6-7) and in fusion with ABD094 (SEQ IDNO:759) via a GS(G₄S)₂ linker (lanes 2-5).

FIGS. 6A and B are diagrams showing exemplary data of dose-responsecharacterization of the potency of different C5 binding Z variants toinhibit complement activation as seen in a hemolytic assay, described inExample 6. C5 deficient serum was diluted 63-fold and supplemented with0.1 nM hC5. A) shows effect of different Z-ABD fusion proteins (Zvariants corresponding to SEQ ID NO:745, SEQ ID NO:748-753 and SEQ IDNO:756 fused to ABD094 (SEQ ID NO:759) by a GS linker) to hC5, whereasB) shows effect of different C5 binding constructs comprising the sameC5 binding Z variant (Z06175a, SEQ ID NO:753) as monomer or dimer, infusion with ABD094 (SEQ ID NO:759), or as provided with a His₆-tag (sixhistidine residues), compared to the C5 binding tick protein OmCI (SEQID NO:761).

FIGS. 7A and B are diagrams showing exemplary data of equilibriumbinding based on the displacement ECL technique described in Example 6.FIG. 7A shows C5 binding of different Z variants (SEQ ID NO:745, SEQ IDNO:748-757) in fusion with ABD094 (SEQ ID NO:759) compared to C5 bindingof the tick protein OmCI (SEQ ID NO:761). FIG. 7B shows binding ofdifferent C5 binding constructs comprising the same C5 binding Z variant(SEQ ID NO:753) as monomer or dimer, in fusion with ABD094 (SEQ IDNO:759) or as provided with a His₆-tag.

FIGS. 8A and 8B show interactions between Z-ABD variants and human serumalbumin (HSA) studied as described in Example 7. A) Size exclusionchromatography (SEC) where Z-ABD (Z06175a (SEQ ID NO:753) fused toABD094 (SEQ ID NO:759) by a GS linker) has been preincubated withequimolar amounts of HSA (1). As a comparison, the chromatograms for HSAalone (2) and Z-ABD alone (3) are also shown in the graph. B) Biacoresensorgrams of Z-ABD and Z-ABD-Z (Z06175a (SEQ ID NO:753) fused toABD094 (SEQ ID NO:759) by linkers specified in FIG. 4, in construct 2and construct 5, respectively) injected over an HSA coated surface. Eachof the two constructs was injected at a concentration of 25, 100 and 400nM.

FIG. 9 is a diagram showing the pharmacokinetic profiles for the C5binding compounds Z-ABD and Z-ABD-Z (Z06175a, SEQ ID NO:753) fused toABD094 (SEQ ID NO:759) by linkers specified in FIG. 4, in construct 2and construct 5, respectively) in Male Sprague Dawley rats over timeafter intravenous (i.v., 0.25 μmol/kg) and subcutaneous (s.c., 0.5μmol/kg) administration, as described in Example 8. Each data pointrepresents an average from three individual animals at a specific timepoint ranging from five minutes to two weeks after dosing for animalsdosed i.v and from 15 minutes to two weeks for animals dosed s.c.

FIG. 10 shows ex vivo hemolysis in sheep erythrocytes after exposure toserum diluted 1:5 from animal samples taken from Sprague Dawley ratsafter intravenous (i.v.; 0.25 μmol/kg) administration of Z-ABD (Z06175a(SEQ ID NO:753) fused to ABD094 (SEQ ID NO:759) by a GS linker), asdescribed in Example 8. Each dot represents one individual animal at aspecific time point ranging from five minutes to two weeks after dosing.

FIG. 11 shows ex vivo hemolysis in sheep erythrocytes after exposure toserum diluted 1:5 from animal samples taken from Sprague Dawley ratsafter subcutaneous (s.c.; 0.5 μmol/kg) administration of Z-ABD (Z06175a(SEQ ID NO:753) fused to ABD094 (SEQ ID NO:759) by a GS linker), asdescribed in Example 8. Each dot represents one individual animal at aspecific time point ranging from 15 minutes to two weeks.

FIG. 12 shows the hemolysis versus Z-ABD (Z06175a (SEQ ID NO:753) fusedto ABD094 (SEQ ID NO:759) by a GS linker) serum concentration followingi.v. and s.c. administration to male Sprague Dawley rats, as describedin Example 8.

FIG. 13 shows hemolysis in sheep erythrocytes versus Z-ABD-Z (Z06175a(SEQ ID NO:753) fused to ABD094 (SEQ ID NO:759) by linkers specified inFIG. 4, construct 5) serum concentration following i.v. and s.c.administration to male Sprague Dawley rats, as described in Example 8.

FIG. 14 shows the serum exposure of Z-ABD (Z06175a (SEQ ID NO:753) fusedto ABD094 (SEQ ID NO:759) by a GS linker) following i.v. (415 nmol/kg)and s.c. (1250 nmol/kg) administration in male Cynomolgus monkey, asdescribed in Example 9. Each data point represents the mean of threeindividual animals.

FIG. 15 is a diagram showing the effect (C5a concentration in lavage) ofthe pro-inflammatory molecule zymosan (40 mg/kg i.p.) alone and incombination with a C5 binding Z-ABD fusion molecule (Z06175a (SEQ IDNO:753) fused to ABD094 (SEQ ID NO:759) by a GS linker)) or OmCI (SEQ IDNO:761) analyzed as described in Example 10. Z-ABD was administered at20 nmol/kg (LD), 100 nmol/kg (MD) and 500 nmol/kg (HD) s.c. 18 h beforeinduction with zymosan. OmCI (30 nmol/kg) was administered i.p. 1 hbefore zymosan treatment and samples were taken 1 h after zymosaninduction.

FIGS. 16A and 16B show the pharmacokinetic profile of Z-ABD (Z06175a(SEQ ID NO:753) fused to ABD094 (SEQ ID NO:759) by a GS linker)following intratracheal administration of 500 nmol/kg into female C57b1mice, as described in Example 11. A) serum concentration in each animal(n=3 for each time point, 27 animals totally) and B) hemolysis in sheeperythrocytes exposed to these serum samples diluted 1:5.

EXAMPLES

The following materials were used throughout this work except whereotherwise noted.

-   -   Escherichia coli strain RR1ΔM15 (Rüther, Nucleic Acids Res        10:5765-5772, 1982).    -   Escherichia coli strain XL1-Blue (Agilent Technologies, cat. no.        200268).    -   Human complement protein C5 (hC5). The full 1676 amino acid        pro-protein has GenBank accession number: NP_001726 (SEQ ID        NO:760) wherein amino acids 19-673 is the beta chain and amino        acids 678-1676 is the alpha chain. Human C5 used herein was        purchased from Quidel (cat. no. A403)    -   Cynomolgus complement protein C5 (cC5; SEQ ID NO:762). The cC5        sequence was derived from the Cynomolgus (Macaca fascicularis)        genomic sequence (www.ebi.ac.uk/ena; Ebeling et al. (2001)        Genome Res. 21(10):1746-1756). The coding region of human C5 was        retrieved from www.ensembl.org, and the C5 gene was localized to        chromosome 15. The region containing the gene is approximately        110 000 bases long and is contained in contigs CAEC01154150 to        CAEC01154178. The contigs were manually joined to a single file        and used as a genomic context for the sim4 software to align the        coding region of human C5 to the raw Cynomolgus genomic        material. Cynomolgus C5 used herein was purified in-house from        serum using a three-step procedure; PEG6000 precipitation, ion        exchange and OmCI affinity chromatography.    -   Rat Complement protein C5 (rC5; GenBank accession number:        XP_001079130) Rat C5 used herein was purified in-house from        serum using a three-step procedure; PEG6000 precipitation, ion        exchange and OmCI affinity chromatography.    -   Human MG7 (hMG7) domain of complement protein C5, corresponding        to amino acid residues 822-931 of human C5 (SEQ ID NO:760;        Fredslund et al. (2008) Nature Immunology 9: 753-760) produced        in-house in Freestyle HEK293 cells.    -   hMG7 binding protein.    -   OmCI (AF2999, Nunn, M. A. et al. supra) from soft tick        Ornithodoros moubata OmCI with a His₆ tag in the C-terminus (SEQ        ID NO:761) was produced in-house in E. coli strain Origami(DE3)        and purified on a HisTrap1 column.

Example 1: Selection and Screening of Complement Protein C5 BindingPolypeptides

Materials and Methods

Biotinylation of Target Protein hC5:

hC5 was biotinylated according to the manufacturer's recommendations atroom temperature (RT) for 40 min using No-Weigh EZ-LinkSulfo-NHS-LC-Biotin (Pierce, cat. no. 21327) at a ten times (10×) molarexcess. Subsequent buffer exchange to PBS (10 mM phosphate, 137 mM NaCl,2.68 mM KCl, pH 7.4) was performed using Protein Desalting Spin Columns(Pierce, cat. no. 89849) according to the manufacturer's instructions.

Phage Display Selection of C5-Binding Polypeptides:

Libraries of random variants of protein Z displayed on bacteriophage,constructed in phagemid pAffi1/pAY00065/pAY02947/pAY02592 essentially asdescribed in Grönwall et al. J Biotechnol 2007, 128:162-183), were usedto select C5 binding polypeptides. Three different library vectors wereused. Two of these utilize an albumin binding domain (ABD, GA3 ofprotein G from Streptococcus strain G148) as fusion partner to the Zvariants generating the libraries Zlib003Naive.I and Zlib006Naive.II.The third library, Zlib004Naive.I utilizes the Taq DNA polymerasebinding molecule Z03639 (denoted Z_(TaqS1-1) in Gunneriusson et al.Protein Eng 1999, 12:873-878) as fusion partner. The libraries had thefollowing actual sizes: 3×10⁹ (Zlib003Naive.I); 1.5×10¹⁰(Zlib006Naive.II); and 1.4×10¹⁰ (Zlib004Naive.I), the number referringto the amount of variants.

Phage stocks were prepared either in shake flasks (Zlib003Naive.I) asdescribed in Grönwall et al. supra or in a 20 l fermenter(Zlib006Naive.II and Zlib004Naive.I). Cells from a glycerol stockcontaining the phagemid library Zlib004Naive.I were inoculated in 20 lof TSB-YE (Tryptic Soy Broth-Yeast Extract; 30 g/l TSB, 5 g/l yeastextract) supplemented with 2% glucose and 100 μg/ml ampicillin. Cellsfrom a glycerol stock containing the phagemid library Zlib006Naive.IIwere inoculated in 20 l of a defined proline free medium [dipotassiumhydrogenphosphate 7 g/l, trisodium citrate dihydrate 1 g/l, uracil 0.02g/l, YNB (Difco™ Yeast Nitrogen Base w/o amino acids, Becton Dickinson)6.7 g/l, glucose monohydrate 5.5 g/l, L-alanine 0.3 g/l, L-argininemonohydrochloride 0.24 g/l, L-asparagine monohydrate 0.11 g/l,L-cysteine 0.1 g/l, L-glutamic acid 0.3 g/l, L-glutamine 0.1 g/l,glycine 0.2 g/l, L-histidine 0.05 g/l, L-isoleucine 0.1 g/l, L-leucine0.1 g/l, L-lysine monohydrochloride 0.25 g/l, L-methionine 0.1 g/l,L-phenylalanine 0.2 g/l, L-serine 0.3 g/l, L-threonine 0.2 g/l,L-tryptophane 0.1 g/l, L-tyrosine 0.05 g/l, L-valine 0.1 g/l]supplemented with 100 μg/ml ampicillin. The cultivations were grown at37° C. in a fermenter (Belach Bioteknik, BR20). When the cells reachedan optical density (OD) of 0.7-0.8, approximately 2.6 l of thecultivation was infected using a 10× molar excess of M13K07 helper phage(New England Biolabs #N03155). The cells were incubated for 30 minutes,whereupon the fermenter were filled up to 20 l with TSB-YE supplementedwith 100 μM IPTG (isopropyl-β-D-1-thiogalactopyranoside, for inductionof expression), 25 μg/ml kanamycin and 12.5 μg/ml carbenicillin andgrown at 30° C. for 22 h. The cells in the cultivation were pelleted bycentrifugation at 15,900 g and the phage particles remaining in themedium were thereafter precipitated twice in PEG/NaCl (polyethyleneglycol/sodium chloride), filtered and dissolved in PBS and glycerol asdescribed in Grönwall et al. supra. Phage stocks were stored at −80° C.before use.

Selections were performed in four cycles against biotinylated hC5. Phagestock preparation, selection procedure and amplification of phagebetween selection cycles were performed essentially as described in WO2009/077175. PBS supplemented with 3% bovine serum albumin (BSA) and0.1% Tween20 was used as selection buffer and the target-phage complexeswere directly captured by DYNABEADS M-280 Streptavidin (Dynal, cat. no.112.06). 1 mg beads per 10 μg complement protein C5 was used. E. colistrain RR1ΔM15 was used for phage amplification. In cycle 1 of theselections, 100 nM hC5 was used and two washes with PBST 0.1% (PBSsupplemented with 0.1% Tween-20) were performed. An increasedstringency, using a lowered target concentration and an increased numberof washes, was applied in the subsequent three cycles. In cycle 2, 3 and4; 50 or 33 nM hC5, 25 or 11 nM hC5 and 12.5 or 3.7 nM hC5 were used. Incycle 2, 3 and 4; 4, 6 and 8 washes were performed, using PBST 0.1% inall cycles or PBST 0.2%, 0.3% and 0.4% in cycle 2, 3 and 4.

ELISA Screening of Z Variants:

To test if the selected Z variant molecules could indeed interact withhuman complement protein C5, ELISA assays were performed. The Z variantswere produced by inoculating single colonies from the selections into 1ml TSB-YE medium supplemented with 100 μg/ml ampicillin and 0.1 mM IPTGin deep-well plates (Nunc, cat. no. 278752). The plates were incubatedfor 18-24 h at 37° C. Cells were pelleted by centrifugation,re-suspended in 300 μl PBST 0.05% and frozen at −80° C. to release theperiplasmic fraction of the cells. Frozen samples were thawed in a waterbath and cells were pelleted by centrifugation. The periplasmicsupernatant contained the Z variants as fusions to an albumin bindingdomain (GM of protein G from Streptococcus strain G148), expressed asAQHDEALE-[Z#####]-VDYV-[ABD]-YVPG (SEQ ID NO. 767) (Grönwall et al,supra), or to the Taq DNA polymerase binding molecule Z03639, expressedas AQHDEALE-[Z#####]-VDYV-[Z03639]-YVPG (SEQ ID NO. 768). Z##### refersto individual 58 amino acid residues Z variants.

Half-area 96-well ELISA plates (Costar, cat. no. 3690) were coated with50 μl/well of coating buffer (50 mM sodium carbonate, pH 9.6) containing4 μg/ml of an antibody specific for Z variants (Affibody, cat. no.20.1000.01.0005) and incubated over-night at 4° C. The antibody solutionwas poured off and the wells were blocked with 100 μl of PBSC (PBSsupplemented with 0.5% casein (Sigma, cat. no. C8654) for 1-2 h at RT.The blocking solution was discarded and 50 μl periplasmic solution wasadded to the wells and incubated for 1 h at RT under slow shaking. Thesupernatants were poured off and the wells were washed 4 times with PBST0.05%. Then 50 μl of biotinylated complement protein hC5, at aconcentration of 5 μg/ml in PBSC, was added to each well. The plateswere incubated for 1.5 h at RT followed by washes as described above.Streptavidin-HRP (Horseradish peroxidase; Dako, cat. no. P0397) wasdiluted 1:10,000 in PBSC, added to the wells which were then incubatedfor 45 min. After washing as described above, 50 μl ImmunoPure TMBsubstrate (Thermo Scientific, cat. no. 34021) was added to the wells andthe plates were treated according to the manufacturer's recommendations.Absorbance of the wells was measured at 450 nm using a multi-well platereader, Victor³ (Perkin Elmer).

As positive control, a periplasmic fraction also containing the PSMAbinding molecule Z03938 expressed asAQHDEALE-[Z03938]-VDYV-[Z03639]-YVPG (SEQ ID NO. 769) was assayedagainst 5 μg/ml biotinylated PSMA protein. As negative control; the sameperiplasmic preparation was assayed against complement protein hC5.Sequencing was performed for the clones with positive absorbance valuesagainst hC5.

Sequencing:

PCR fragments were amplified from single colonies using a standard PCRprogram and the primers AFFI-21 (5′-tgcttccggctcgtatgttgtgtg) (SEQ IDNO. 770) and AFFI-22 (5′-cggaaccagagccaccaccgg) (SEQ ID NO. 771).Sequencing of amplified fragments was performed using the biotinylatedoligonucleotide AFFI-72 (5′-biotin-cggaaccagagccaccaccgg) (SEQ ID NO.772) and a BIGDYE Terminator v3.1 Cycle Sequencing Kit (AppliedBiosystems), used in accordance with the manufacturer's protocol. Thesequencing reactions were purified by binding to magnetic streptavidincoated beads (Detach Streptavidin Beads, Nordiag, cat. no. 2012-01)using a Magnatrix 8000 (Magnetic Biosolution), and analyzed on ABI PRISM3100 Genetic Analyzer (PE Applied Biosystems).

Blocking ELISA:

Clones found positive for hC5 in the ELISA screening were subjected toan ELISA blocking assay in order to elucidate if their target bindingwas affected by the presence of the hC5 binding proteins OmCI and/orhMG7 binding protein. The blocking ELISA was run using Z variantsexpressed in periplasmic fractions as described in the section for ELISAscreening above, but setting up 5 ml cultures in 12 ml round-bottomtubes and using 2 ml PBST 0.05% for pellet dissolution. The ELISAblocking assay was run as the ELISA screening assay, with a protocolmodification introduced at the target step; OmCI or hMG7 binding proteinwere mixed with the target protein before addition to the assay plate. 5μg/ml biotinylated hC5 was mixed with 5 times or 20 times molar excessof OmCI or hMG7 binding protein, respectively, then incubated 1 h at RTto allow complex formation before addition to the plate. For each clone,a reference (1), a negative control (2) and a background (3)response/signal, respectively, were obtained as follows: at the targetstep, solely hC5 was added to the Z variants (as in the screening ELISA)(1); the irrelevant protein PSMA (in house produced) was added tocomplement protein hC5, instead of OmCI or hMG7 binding protein (2);only buffer was added to the Z variants (3).

Results

Phage Display Selection of Complement Protein C5 Binding Polypeptides:

Individual clones were obtained after two-four cycles of phage displayselections against biotinylated hC5.

ELISA Screening of Z Variants:

The clones obtained after four cycles of selection were produced in96-well plates and screened for complement protein C5 binding activityin ELISA. In total, nearly 400 clones were screened. The absorbancemeasurements showed many clearly hC5 positive clones. The result from aselection of clones is displayed in Table 1; the Z05363 (SEQ ID NO:510)variant is tagged with ABD, whereas the other listed Z variants aretagged with the Taq binding molecule Z03639 as described in the methodssection. The PSMA specific molecule Z03938 used as a negative controlgave a positive signal for PSMA, whereas no signal was obtained againsthC5.

Blocking ELISA:

Clones positive for hC5 were subjected to a blocking assay using the hC5binding proteins OmCI and hMG7 binding protein. For five clones, thebinding signal to complement protein C5 was completely extinguished bythe presence of OmCI, reaching the same level as the background (Table1). One of these clones, namely the Z05363 variant (SEQ ID NO:510), wasalso tested for its ability to bind hC5 in the presence of hMG7 bindingprotein. The hMG7 binding protein did not inhibit the binding of Z05363to hC5.

TABLE 1 Response in ELISA to target, with or without blocking moleculefor a number of Z variants. Z variant SEQ ID NO: # hC5 (OD 450 nm)OmCI-block Z05363 SEQ ID NO: 510 3.143 complete Z05477 SEQ ID NO: 5092.872 complete Z05483 SEQ ID NO: 511 0.531 complete Z05538 SEQ ID NO:512 0.099 complete Z05692 SEQ ID NO: 513 0.944 completeSequencing:

Sequencing was performed for the clones with positive absorbance valuesagainst complement protein C5 in the ELISA screening. Each variant wasgiven a unique identification number #####, and individual variants arereferred to as Z#####. The amino acid sequences of the 58 amino acidresidues long Z variants are listed in FIG. 1 and in the sequencelisting as SEQ ID NO:509-513. The deduced complement protein C5 bindingmotifs of these Z variants are listed in FIG. 1 and in the sequencelisting as SEQ ID NO:13-17. The amino acid sequences of the 49 aminoacid residues long polypeptides predicted to constitute the completethree-helix bundle within each of these Z variants are listed in FIG. 1and in the sequence listing as SEQ ID NO:261-265.

Example 2: Production and Characterization of Z Variants

Materials and Methods

Subcloning of Z Variants, Protein Expression and Purification:

Five complement protein C5 binding Z variants (Z05363 (SEQ ID NO:510);Z05477 (SEQ ID NO:509); Z05483 (SEQ ID NO:511); Z05538 (SEQ ID NO:512)and Z05692 (SEQ ID NO:513)) were amplified from pAffi1/pAY00065/pAY02947library vectors. A subcloning strategy for construction of dimericAffibody molecules with N-terminal His₆ tags was applied using standardmolecular biology techniques and as described in detail in WO2009/077175. The Z gene fragments were subcloned into the expressionvector pAY01448 resulting in the encoded sequenceMGSSHHHHHHLQ-[Z#####][Z#####]-VD (SEQ ID NO. 773).

The subcloned Z variants were transformed into E. coli BL21(DE3) andexpressed in the multifermenter system Greta (Belach Bioteknik). Inbrief, cultures were grown at 37° C. in 800 ml TSB-YE-medium containing50 μg/ml kanamycin. At an OD₆₀₀ of ˜1, the cultures were induced throughthe automatic addition of IPTG to a final concentration of 0.05 mM.Cultures were cooled down to approximately 10° C. after 5 h ofinduction, and harvested by centrifugation (20 min, 15,900 g).Supernatants were discarded and the cell pellets were collected andstored at −20° C. until further use. Expression levels and the degree ofsolubility were estimated by SDS-PAGE analysis on 4-12% NUPAGE gels(Invitrogen) using Coomassie blue staining.

For Z variants expressed mainly as soluble protein, the cell pelletswere resuspended in binding buffer (20 mM sodium phosphate, 0.5 M NaCl,20 mM imidazole, pH 7.4) with an addition of 1000 U BENZONASE (Merck,cat. no. 1.01654.001) and disrupted by ultrasonication. For each of theZ variants, the sonicated suspension was clarified by centrifugation (40min, 25,000 g, 4° C.) and the supernatant was loaded onto a 1 ml HisGRAVITRAP column (GE Healthcare). The column was washed with wash buffer(20 mM sodium phosphate, 0.5 M NaCl, 60 mM imidazole, pH 7.4), beforeeluting the Z variants with 3 ml elution buffer (20 mM sodium phosphate,0.5 M NaCl, 0.5 M imidazole, pH 7.4). Z variants which expressed mainlyas insoluble protein were purified likewise, but 8 M urea was includedin the binding and wash buffer. If required, the Z variants were furtherpurified by reversed phase chromatography (RPC) on 1 ml RESOURCE columns(GE Healthcare) using water including 0.1% TFA (trifluoroacetic acid) asmobile phase and elution with an appropriate gradient (typically 0-50%over 20 column volumes) of acetonitrile including 0.1% TFA.

The buffer was exchanged to PBS using PD-10 columns (GE Healthcare).

Protein Characterization:

The concentration of the purified Z variants was determined byabsorbance measurements at 280 nm using theoretical extinctioncoefficients. The purity was estimated by SDS-PAGE analysis on 4-12%NUPAGE gels (Invitrogen) using Coomassie blue staining. To verify theidentity and to determine the molecular weights of purified Z variants,LC/MS-analyses were performed on an Agilent 1100 LC/MSD system (AgilentTechnologies).

CD Analysis:

The purified Z variants were diluted to 0.5 mg/ml in PBS. For eachdiluted Z variant, a CD spectrum was recorded between 250-195 nm at atemperature of 20° C. In addition, a variable temperature measurement(VTM) was performed to determine the melting temperature (Tm). In theVTM, the absorbance was measured at 221 nm while the temperature wasraised from 20 to 90° C., with a temperature slope of 5° C./min. Theability of the Z variant to refold was assessed by collecting anadditional CD spectrum at 250-195 nm after cooling to 20° C. The CDmeasurements were performed on a Jasco J-810 spectropolarimeter (JascoScandinavia AB) using a cell with an optical path length of 1 mm.

Biacore Binding Analysis:

The interactions of the five subcloned His₆-tagged dimeric hC5-binding Zvariants with hC5, cC5, rC5, hMG7 and hIgG (Sigma, cat. no. G4386) wereanalyzed in a Biacore instrument (GE Healthcare). The Z variants wereimmobilized in different flow cells on the carboxylated dextran layer ofseveral CM5 chip surfaces (GE Healthcare). The immobilization wasperformed using amine coupling chemistry according to the manufacturer'sprotocol. One flow cell surface on each chip was activated anddeactivated for use as blank during analyte injections. The analytes,diluted in HBS-EP running buffer (GE Healthcare) to a finalconcentration of 100 nM, were injected at a flow rate of 10 μl/min for 1min. After 2 min of dissociation, the surfaces were regenerated with oneinjection of 10 mM HCl. The results were analyzed in BiaEvaluationsoftware (GE Healthcare). Curves of the blank surface were subtractedfrom the curves of the ligand surfaces.

Results

Subcloning of Z Variants:

Five selected unique clones (Z05477 (SEQ ID NO:509), Z05363 (SEQ IDNO:510), Z05483 (SEQ ID NO:511), Z05538 (SEQ ID NO:512) and Z05692 (SEQID NO:513)) were chosen for subcloning as dimers in the expressionvector pAY01448 and were subsequently verified by sequencing.

Protein Production:

The histidine-tagged dimeric Z variants yielded acceptable expressionlevels of soluble gene product. The purity of produced batches wasestimated to exceed 90% as assessed by SDS-PAGE analysis. LC/MS analysisverified the correct molecular weight for all Z variant molecules.

CD Analysis:

The melting temperatures (Tm) of the different Z variants werecalculated by determining the midpoint of the transition in the CDsignal vs. temperature plot. The results for a number of reversiblyfolding Z variants are summarized in Table 2 below.

TABLE 2 Melting temperatures for a number of Z variants. SEQ ID NO: # ofTm Z variant monomeric Z variant (° C.) His₆-(Z05477)₂ SEQ ID NO: 509 45His₆-(Z05363)₂ SEQ ID NO: 510 35 His₆-(Z05483)₂ SEQ ID NO: 511 44His₆-(Z05538)₂ SEQ ID NO: 512 54 His₆-(Z05692)₂ SEQ ID NO: 513 52Biacore Binding Analysis:

The binding of the five subcloned dimeric Z variants to differentspecies of C5 and MG7, a subdomain of hC5, as well as the backgroundbinding to IgG was tested in a Biacore instrument by injecting thedifferent proteins over surfaces containing the Z variants. The ligandimmobilization levels for the different Z variants on the surfaces were:Z05363: 2080 RU, Z05477: 2180 RU, Z05483: 2010 RU, Z05538: 2570 RU andZ05692: 3270 RU. The different Z variants were tested for binding todifferent sets of proteins injected at concentrations of 100 nM, seeTable 3. The result for the tested Z variants is displayed in the tableas a +/− outcome for each protein. As an example of the Biacore bindinganalysis, FIG. 2 shows the sensorgrams obtained from immobilized dimericZ05477 assayed against hC5, cC5, rC5, hMG7 and hIgG.

TABLE 3 Biacore response of different Z variants against C5 from variousspecies and relevant selected background proteins. SEQ ID NO: # of Zvariant monomeric Z variant hC5 cC5 rC5 hMG7 hIgG His₆-(Z05477)₂ SEQ IDNO: 509 + + + − − His₆-(Z05363)₂ SEQ ID NO: 510 + + + − − His₆-(Z05483)₂SEQ ID NO: 511 + + + − − His₆-(Z05538)₂ SEQ ID NO: 512 + + + − −His₆-(Z05692)₂ SEQ ID NO: 513 + + − − −

Example 3: Design and Construction of a Maturated Library of ComplementProtein C5 Binding Z Variants

In this Example, a maturated library was constructed. The library wasused for selections of hC5-binding polypeptides. Selections frommaturated libraries are usually expected to result in binders withincreased affinity (Orlova et al. Cancer Res 2006, 66(8):4339-48). Inthis study, randomized double stranded linkers were generated by theSLONOMICS technology which enables incorporation of randomized sets oftrinucleotide building blocks using ligations and restrictions of thesubsequently built up double stranded DNA.

Materials and Methods

Library Design:

The library was based on a selection of sequences of the hC5 binding Zvariants described in Examples 1 and 2. In the new library, 13 variablepositions in the Z molecule scaffold were biased towards certain aminoacid residues, according to a strategy based on the Z variant sequencesdefined in SEQ ID NO:509-513 (Z05477, Z05363, Z05483, Z05538, Z05692). ASLONOMAX library of double-stranded DNA, containing the 147 bp partiallyrandomized helix 1 and 2 of the amino acid sequence 5′-AA ATA AAT CTCGAG GTA GAT GCC AAA TAC GCC AAA GAA/GAG NNN NNN NNN GCA/GCC NNN NNNGAG/GAA ATC/ATT NNN NNN TTA/CTG CCT AAC TTA ACC/ACT NNN NNN CAA/CAG TGGNNN GCC/GCG TTC ATC/ATT NNN AAA/AAG TTA/CTG NNN GAT/GAC GAC CCA AGC CAGAGC TCA TTA TTT A-3′ (SEQ ID NO. 774) (randomized codons are illustratedas NNN) flanked with restriction sites XhoI and SacI, was ordered fromSloning BioTechnology GmbH (Pucheim, Germany). The theoreticaldistributions of amino acid residues in the new library finallyincluding 12 variable Z positions are given in Table 4.

TABLE 4 Library design. Amino acid position No in the Z of variantRandomization amino Pro- molecule (amino acid abbreviations) acidsportion 9 H, Q, S, T, V  5 1/5  10 I, L, V, W  4 1/4  11 A, D, E, H, K,L, N, R, S, T, Y 12 1/12 13 N, Q, W, Y  4 1/4  14 A, D, E, H, I, K, L,N, Q, R, S, T, V, W, Y 15 1/14 17 D, E  2 1/2  18 A, D, E, G, H, I, K,L, Q, R, S, T, V, Y 14 1/14 24 I, L, V  3 1/3  25 A, D, E, H, K, N, Q,R, S, T, Y 11 1/11 28 I, L, V  3 1/3  32 A, D, E, F, G, H, K, L, N, Q,R, S, T, V 14 1/14 35 A, D, E, H, K, N, Q, R, S, T, W, Y 12 1/12Library Construction:

The library was amplified using AmpliTaq Gold polymerase (AppliedBiosystems, cat. no. 4311816) during 12 cycles of PCR and pooledproducts were purified with QIAquick PCR Purification Kit (QIAGEN, cat.no. 28106) according to the supplier's recommendations. The purifiedpool of randomized library fragments was digested with restrictionenzymes XhoI and SacI (New England Biolabs, cat. no. R01460L, and cat.no. R0156L) and purified once more with PCR Purification Kit.Subsequently, the product was purified using preparative gelelectrophoresis on a 1% agarose gel.

The phagemid vector pAY02592 (essentially as pAffi1 described inGrönwall et al. supra) was restricted with the same enzymes, purifiedusing phenol/chloroform extraction and ethanol precipitation. Therestricted fragments and the restricted vector were ligated in a molarratio of 5:1 with T4 DNA ligase (New England Biolabs, cat. no. M0202S),for 2 hours at RT followed by overnight incubation at 4° C. The ligatedDNA was recovered by phenol/chloroform extraction and ethanolprecipitation, followed by dissolution in 10 mM Tris-HCl, pH 8.5.

The ligation reactions (approximately 250 ng DNA/transformation) wereelectroporated into electrocompetent E. coli RR1ΔM15 cells (100 μl).Immediately after electroporation, approximately 1 ml of SOC medium(TSB-YE media, 1% glucose, 50 μM MgCl₂, 50 μM MgSO₄, 50 μM NaCl and 12.5μM KCl) was added. The transformed cells were incubated at 37° C. for 50min Samples were taken for titration and for determination of the numberof transformants. The cells were thereafter pooled and cultivatedovernight at 37° C. in 71 of TSB-YE medium, supplemented with 2% glucoseand 100 μg/ml ampicillin. The cells were pelleted for 15 min at 4,000 g,resuspended in a PBS/glycerol solution (approximately 40% glycerol). Thecells were aliquoted and stored at −80° C. Clones from the library of Zvariants were sequenced in order to verify the content and to evaluatethe outcome of the constructed library vis-à-vis the library design.Sequencing was performed as described in Example 1 and the amino aciddistribution was verified.

Preparation of Phage Stock:

Cells from the glycerol stock containing the C5 phagemid library wereinoculated in 20 l of a defined proline free medium (described inExample 1) supplemented with 100 μg/ml ampicillin, and grown at 37° C.in a fermenter (Belach Bioteknik, BR20). All steps were performed asdescribed in Example 1 for the library Zlib006Naive.II. Aftercultivation, the cells were pelleted by centrifugation at 15,900 g andthe phage particles remaining in the medium were thereafter precipitatedtwice in PEG/NaCl, filtered and dissolved in PBS and glycerol asdescribed in Example 1. Phage stocks were stored at −80° C. until use inselection.

Results

Library Construction:

The new library was designed based on a set of OmCI-blocked C5 binding Zvariants with verified binding properties (Example 1 and 2). Thetheoretical size of the designed library was 6.7×10⁹ Z variants. Theactual size of the library, determined by titration after transformationto E. coli. RR1ΔM15 cells, was 1.4×10⁹ transformants.

The library quality was tested by sequencing of 64 transformants and bycomparing their actual sequences with the theoretical design. Thecontents of the actual library compared to the designed library wereshown to be satisfying. The locked position in the designed amino acidsequence (W in position 27) was reflected in the actual sequence in thatonly the expected amino acid occurred in that position. A maturatedlibrary of hC5 binding polypeptides was thus successfully constructed.

Example 4: Selection, Screening and Characterization of Z Variants froma Maturated Library

Materials and Methods

Phage Display Selection of Complement Protein C5 Binding Polypeptides:

The target protein hC5 was biotinylated as described in Example 1. Phagedisplay selections were performed against hC5 essentially as describedin Example 1 using the new library of Z variant molecules described inExample 3. E. coli XL1-Blue was used for phage amplification. Selectionwas initially performed in two parallel tracks. In one track, the timeof selection was 2 h, while in the other track, shorter selection timeswere used: 20 min in the first cycle and 10 min for subsequent cycles2-4. These two tracks (1 and 2) were further divided in the secondcycle, resulting in totally six tracks (1a-c and 2a-c, differing intarget concentration and wash conditions). Selection was performed in atotal of four cycles. In cycle 1 of the selections, 25 nM complementprotein C5 was used and five washes with PBST 0.1% were performed. Anincreased stringency, using a lowered target concentration and anincreased number of washes, was applied in the subsequent three cycles.In cycle 2, 3 and 4; 10, 5 or 2.5 nM complement protein C5, 4, 1 or 0.25nM complement protein C5 and 1.6, 0.2 or 0.05 nM complement protein C5were used. In cycle 2, 3 and 4; 10, 15 and 20 washes were performedusing PBST 0.1%. In addition, the second last wash was prolonged to 3 hwith a 50× excess of non-biotinylated hC5 in the washing solution fortwo of the tracks (1c and 2c).

Sequencing of Potential Binders:

Individual clones from the different selection tracks were picked forsequencing. All clones run in the ELISA screening were sequenced.Amplification of gene fragments and sequence analysis of gene fragmentswere performed as described in Example 1.

ELISA Screening of Z Variants:

Single colonies containing Z variants were randomly picked from theselected clones of the complement protein C5 maturated library and grownin 1 ml cultivations as described in Example 1. Periplasmic proteinswere released by 8 repeated freeze-thawing cycles. ELISA screenings wereperformed essentially as described in Example 1 with the followingexceptions. Half-area 96-well ELISA plates were coated with 2 μg/ml ofan ABD specific goat antibody (in house produced) diluted in coatingbuffer. Biotinylated hC5 was used at a concentration of 0.15 μg/ml andincubation performed for 1.5-2 h. Streptavidin conjugated HRP wasobtained from Thermo Scientific (cat. no. N100). The Z variant Z05363(SEQ ID NO:510) originating from the primary selections (Example 1) wasused as a positive control as well as a negative control omitting hC5.

Selected maturated Z variants were subjected to a second screen againsthC5 at a lower concentration and compared to rC5. The assay wasessentially performed as described above. hC5 and rC5 was used at aconcentration of 0.05 μg/ml and 4 μg/ml, respectively. The Z variantZ05363 (SEQ ID NO:510) was used as a positive control in this experimentas well. As a negative control, a Z variant binding to PDGF-Rβ (Z01977;described in WO 2009/077175) was assayed against biotinylated hC5 orrC5.

In deep sequence analysis of selected Z variants and correlation ofamino acids in the 13 randomized positions with measured meltingtemperatures and IC₅₀ values for human C5 and mouse C5 in the hemolysisassay (described in Example 6) suggested a favorable Z variant notidentified among the 558 sequenced clones. Based on the Z variant Z05998(SEQ ID No:499), a single amino acid, Ile in position 10 was substitutedwith Leu using conventional technology for site directed mutagenesis.The new variant is referred to as Z08044 (SEQ ID NO:498). The deducedcomplement protein C5 binding motif of this Z variant is listed in FIG.1 and in the sequence listing as SEQ ID NO:2. The amino acid sequencesof the 49 amino acid residues long polypeptide predicted to constitutethe complete three-helix bundle within these Z variant is listed in FIG.1 and in the sequence listing as SEQ ID NO:250.

Results

Phage Display Selection of Complement Protein C5 Binding Polypeptides:

Selection was performed in totally six parallel tracks containing fourcycles each. The different selection tracks differed in targetconcentration and wash conditions as follows: 1a) 2 h selection time,high concentration, standard wash, 1b) 2 h selection time, lowconcentration, standard wash, 1c) 2 h selection time, mediumconcentration, long wash, 2a) 10 min selection time, high concentration,standard wash, 2b) 10 min selection time, low concentration, standardwash, and 2c) 10 min selection time, medium concentration, long wash.For each selection cycle, the target concentration was decreased and thewashing conditions were more stringent. All tracks gave in each roundsufficient amounts of phage particles in the eluate. Most phageparticles were found in tracks 1a and 2a, representing the highesttarget concentration and mildest wash conditions.

Sequencing:

Randomly picked clones (558) were sequenced. Each individual Z variantwas given an identification number, Z#####, as described in Example 1.In total, 242 new unique Z variant molecules were identified. The aminoacid sequences of the 58 amino acid residues long Z variants are listedin FIG. 1 and in the sequence listing as SEQ ID NO:497, SEQ IDNO:499-508 and SEQ ID NO:514-744. The deduced complement protein C5binding motifs of these Z variants are listed in FIG. 1 and in thesequence listing as SEQ ID NO:1, SEQ ID NO:3-12 and SEQ ID NO:18-248.The amino acid sequences of the 49 amino acid residues long polypeptidespredicted to constitute the complete three-helix bundle within each ofthese Z variants are listed in FIG. 1 and in the sequence listing as SEQID NO:249, SEQ ID NO:251-260 and SEQ ID NO:266-496. Among the sequencedclones, 63 sequences occurred two or more times.

ELISA Screening of Z Variants:

Clones obtained after four selection cycles were produced in 96-wellplates and screened for hC5-binding activity using ELISA. All randomlypicked clones were analyzed. 229 of the 242 unique Z variants were foundto give a higher response (0.3-3.1 AU) against hC5 at a concentration of0.15 μg/ml compared to the positive control clone Z05363 (SEQ ID NO:510;an average absorbance signal of 0.3 AU), obtained from the primaryselections (Example 1). Clones from all selection tracks showed positivesignals. The negative controls had an absorbance of approximately 0.1AU.

Z variants were selected based on their performance in the ELISA screenagainst hC5 and the occurrence frequency. 43 unique Z variants wereassayed against a lower concentration of hC5 (0.05 μg/ml) as well as rC5(4 μg/ml). A positive result against rC5 was obtained for 40 of thetested Z variants, defined as 2× the signal for the negative control(0.4 AU). The results for all the tested Z variants against the lowerconcentration of hC5 as well as against rC5 are shown in FIG. 3.

Example 5: Subcloning, Production and Characterization of a Subset ofComplement Protein C5 Binding Z Variants

Materials and Methods

Subcloning of Z Variant Molecules into Expression Vectors:

Based on sequence analysis and the performance in the ELISA againsthuman and rat complement protein C5, 45 clones were selected forsubcloning into the expression vector pAY01448. Monomer Z variantfragments were amplified from the phagemid vector pAY02592 and thesubcloning into pAY01448 was performed as described in Example 2,resulting in a vector encoding the protein sequenceMGSSHHHHHHLQ-[Z#####]-VD (SEQ ID NO. 775).

Protein Expression and Purification:

The 45 Z variants in the His₆-(Z#####) format, were expressed in anautomated multifermenter system as described in Example 2 or similarlyin a small scale set-up of 100 ml cultures in shaker flasks inducedmanually with IPTG to a final concentration of 0.4 mM. Purification wasperformed using 1 ml His GRAVITRAP columns essentially as described inExample 2 or in a smaller scale using 0.1 ml His SpinTrap (GEHealthcare, cat. no. 28-4013-53). Buffer was exchanged to PBS usingPD-10 columns or PD SpinTrap G-25 (GE Healthcare, cat. no. 28-9180-04)according to the manufacturer's instructions. The concentration ofpurified Z variants was determined by absorbance measurements at 280 nmand the purity and identity was assessed by SDS-PAGE and LC/MS asdescribed in Example 2. Samples were aliquoted and stored at −80° C.until further use.

CD Analysis:

The CD analysis for determination of melting temperatures and foldingreversibility was performed as described in Example 2.

Results

Protein Expression and Purification:

All 45 subcloned Z variants could be expressed and the in vitrosolubility for all purified variants was good. The purity was estimatedby LC/MS to exceed 90% for all variants. The correct molecular weightswere verified by LC-MS.

CD Analysis:

CD spectrum measurements performed at 20° C. confirmed the α-helicalstructure of the Z variants at this temperature. An overlay of thespectrums obtained after the variable temperature measurements (heatingto 90° C. followed by cooling to 20° C.) on the spectrums obtainedbefore the variable temperature measurement showed that all Z variantsfold back completely, or nearly completely, to their α-helicalstructures after heating to 90° C. (results not shown). The meltingtemperatures for a set of Z variants were determined from the variabletemperature measurements and are shown in Table 5.

TABLE 5 Melting temperatures of maturated Z variants with a histidinetag fused directly to the amino terminus of SEQ ID NO: 497 and SEQ IDNO: 499-508. Z variant SEQ ID NO: # of Z variant Tm (° C.) His₆-Z06175SEQ ID NO: 497 44 His₆-Z05998 SEQ ID NO: 499 45 His₆-Z06009 SEQ ID NO:500 45 His₆-Z06079 SEQ ID NO: 501 46 His₆-Z06126 SEQ ID NO: 502 44His₆-Z06140 SEQ ID NO: 503 42 His₆-Z06189 SEQ ID NO: 504 47 His₆-Z06214SEQ ID NO: 505 44 His₆-Z06215 SEQ ID NO: 506 41 His₆-Z06226 SEQ ID NO:507 44 His₆-Z06018 SEQ ID NO: 508 46

Example 6: In Vitro Characterization of C5 Binding Z Variants

Materials and Methods

Cloning and Protein Production:

DNA encoding a subset of C5 binding Z variants (SEQ ID NO:745-757) whereE. coli codon optimized and synthesized by GeneArt, GmbH. The syntheticgenes representing the C5 binding Z variants were subcloned andexpressed in E. coli. The expression vectors encoding constructs ofmonomers or dimers of Z variants optionally linked to an albumin bindingdomain (ABD094, SEQ ID NO:759) are schematically illustrated in FIG. 4.

Intracellularly expressed Z variants were purified using conventionalchromatography methods. Homogenization and clarification was performedby sonication followed by centrifugation and filtration. Anion exchangechromatography was used as capture step. Further purification wasobtained by hydrophobic interaction chromatography. The purificationswere executed at acidic conditions (pH 5.5). Polishing and bufferexchange was performed by size exclusion chromatography. Beforeconcentration to final protein content, the endotoxin level was reducedby polymyxin B affinity chromatography. Produced proteins were analyzedby MALDI-TOF MS and on SDS-PAGE.

In addition, recombinantly expressed OmCI protein (SEQ ID NO:761) wasused as a reference molecule in the in vitro studies.

Inhibition of Hemolysis:

For studies of classical complement pathway function and inhibitionthereof by C5 binding polypeptides, sheep erythrocytes were preparedfrom fresh sheep whole blood in Alsever's solution (Swedish NationalVeterinary Institute) and thereafter treated with rabbit anti-sheeperythrocyte antiserum (Sigma) to become antibody sensitized sheeperythrocyte (EA). The whole process was conducted under asepticconditions. All other reagents were from commercial sources.

The in vitro assay was run in 96-well U-form microtiter plate byconsecutive additions of a test protein, a complement serum and EAsuspension. The final concentrations of all reagents, in a totalreaction volume of 50 μl per well and at pH 7.3-7.4, were: 0.15 mMCaCl₂; 0.5 mM MgCl₂; 3 mM NaN₃; 138 mM NaCl; 0.1% gelatin; 1.8 mM sodiumbarbital; 3.1 mM barbituric acid; 5 million EA; complement protein C5serum at suitable dilution, and C5 binding Z variant at desiredconcentrations. Different species of complement sera were used in theassay to define cross-species potencies of the Z variants. For mouseserum, a C5 depleted human serum (C5D from Quidel cat. no. A501) had tobe supplemented in an equal amount.

The Z variants were pre-incubated with the above described complementserum for 20 min on ice prior to starting the reaction by the additionof EA suspension. The hemolytic reaction was allowed to proceed at 37°C. during agitation for 45 min and was then optionally ended by additionof 100 μl ice-cold saline containing 0.02% Tween 20. The cells werecentrifuged to the bottom and the upper portion, corresponding to 100 μlsupernatant, was transferred to a transparent microplate havinghalf-area and flat-bottom wells. The reaction results were analyzed asoptical density using a microtiter plate reader at a wavelength of 415nm.

On all test occasions, controls, vehicle and OmCI (SEQ ID NO:761), wereincluded in each plate to define the values of uninhibited and fullyinhibited reactions, respectively. These values were used to calculatethe % inhibition of the complement hemolysis at any given sampleconcentration. The inhibitory potencies (IC₅₀ values) of tested Zvariants were defined by applying the same assay in the presence of acontrolled concentration of human C5 added to C5 depleted serum. Forhighly potent inhibitors (low nanomolar to sub-nanomolar), a final C5concentration of the reaction mixture was controlled at 0.1 nM, whichwas optionally established by using C5 depleted or deficient sera.

In Vitro Kinetics and Affinity of C5 Binding Z Variants to ImmobilizedhC5:

The binding affinity of a number of C5 binding Z variants (SEQ IDNO:748-757) to hC5 were analyzed using a Biacore T200 instrument (GEHealthcare). Human C5 (A403, Quidel Corporation) was coupled to a CM5sensor chip (900 RU) using amine coupling chemistry according to themanufacturer's protocol. The coupling was performed by injecting hC5 ata concentration of 7.5 μg/ml in 10 mM Na-acetate buffer pH 5 (GEHealthcare). The reference cell was treated with the same reagents butwithout injecting human C5.

All experiments were performed in 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mMEDTA, 0.005% Surfactant P20 (HBS-EP buffer, GE Healthcare). For kineticanalyses, the flow rate was 30 μl/min and data were collected at 25° C.Data from the reference cell were subtracted to compensate for bulkrefractive index changes. In most cases, an injection of HBS-EP was alsoincluded as control so that the sensorgrams were double blanked. Thesurfaces were regenerated in HBS-EP buffer.

Binding of Z variants to immobilized hC5 was studied with the singlecycle kinetics method, in which five concentrations of sample areinjected one after the other in the same cycle without regenerationbetween injections. Kinetic constants were calculated from thesensorgrams using the Langmuir 1:1 or bivalent analyte model of BiacoreT200 Evaluation Software version 1.0.

In Vitro Kinetics and Affinity of C5 Binding Z-ABD Molecules toImmobilized hC5:

Binding of Z-ABD molecules (SEQ ID NO:748-757 fused to ABD094 (SEQ IDNO:759) by a GS linker), to immobilized hC5 was evaluated using aBiacore T200 instrument (GE Healthcare).

Z-ABD constructs where Z06175a (SEQ ID NO:753) as a monomer or dimerhave been fused to ABD094 (SEQ ID NO:759) either in the N-terminus orthe C-terminus via different linkers as specified in FIG. 4 (constructs2, 7, 5 and 4) were also pre-incubated with recombinant human albumin(Cell Prime rAlbumin AF-G, 9301, Novozymes), diluted and then injectedover immobilized human C5 according to the single-cycle kinetics methodas described above. As a comparison, the same constructs were injectedin the absence of HSA. Two constructs, Z06175a-GS (FIG. 4, construct 1)and Z06175a-GSGGGGSGGGGS-ABD094 (SEQ ID NO. 776) (FIG. 4, construct 3)were only tested in the absence of HSA.

Steady State Binding of C5 Binding Z Variants to C5 Coated ECL Plates:

The affinity of a number of C5 binding constructs comprising Z variants(SEQ ID NO:745, SEQ ID NO:748-757 optionally fused to ABD094 (SEQ IDNO:759) in constructs as specified in FIG. 4) to human C5 was measuredby displacement of a ruthenium labeled C5 binding Z-ABD variant (SEQ IDNO:748 fused to SEQ ID NO:759 by a GS-linker).

The Z-ABD variant (SEQ ID NO:748 fused to SEQ ID NO:759 by a GS-linker)to be used as tracer was labeled at a molar ratio 1:12 to 1:20 (protein:SULFO-TAG NHS-Ester, Meso Scale Discovery, cat. no. R91AN-1). Thelabeling reaction was performed on ice for two hours. Unbound SULFO-TAGwas removed using a ZEBA spin desalting column (Thermo Scientific, catno. 89889) and final protein concentration was measured by usingBradford reagent (Bradford, M. M., Anal. Biochem. 72: 248-254, 1976).The affinity (dissociation constant, K_(D)) of the SULFO-TAG labeledZ-ABD variant was determined by saturation binding analysis ofincreasing concentrations of the labeled Z-ABD variant to C5 coatedelectrochemoluminescence wells (ECL, Meso Scale Discovery). The labeledZ-ABD variant was further analyzed by LC/MS in order to determine thedistribution of SULFO-TAG molecules on the Z-ABD variant.

Displacement was carried out by coating ECL, Multi-array 96-wellhigh-bind, non-coated (Meso Scale Discovery, cat. no. L15XB) plates with50 fmol/well hC5 over night at 4° C. Subsequently, non-specific siteswere blocked with PBS with 1% Casein for two hours at RT. Different Zvariants optionally fused with ABD094 (SEQ ID NO:759) (see FIG. 4) wereincubated at different concentrations along with approximately 100 pM ofthe SULFO-TAG labeled C5 binding Z-ABD variant in PBS with 1% Casein.Incubation lasted three hours at RT while agitating the plate at 300rpm. Finally, incubation was terminated by washing 3 times with 150 μlice-cold PBS-Tween20. Immediately after the final wash, 150 μl 2×reading buffer (4× reading buffer T, Meso Scale Discovery cat. no.R92TC-3 diluted 1:1 in ultrapure H₂O) was added to each well and thesignal was detected using a plate reader (SECTOR Imager 2400, Meso ScaleDiscovery). The naturally occurring C5 binding protein OmCI (Nunn et al.supra, SEQ ID NO:761) was included in the displacement assay as apositive control. Binding affinity of competing C5 binding constructsand controls to C5 was determined by non-linear regression analysisusing Excel plugin XLfit5 and GraphPad Prism 4.

Selectivity of Z-ABD Binding to C5 Over C3, C4 and IgG:

Binding of one Z-ABD variant (SEQ ID NO:748 fused to SEQ ID NO:759 by aGS-linker) to the closely related complement proteins C3 and C4 fromhuman as well as binding to human IgG (since the origin of the Z-domain,Staphylococcal protein A, is an IgG binding protein) was addressed bysurface plasmon resonance (SPR) using a Biacore 2000 instrument (GEHealthcare). The Z-ABD construct was immobilized on a CM5 chip(GE-Healthcare) using amine coupling (70 RU). 40 nM and 400 nM of eachof human C3 (A401, Quidel), C4 (A402, Quidel) and IgG (12511, Sigma)diluted in HBS-P buffer (GE Healthcare) were injected over the surface.Each injection was followed by a regeneration cycle with 20 mM NaOHinjected for 30 s. Human C5 at the same concentrations was run inparallel as a positive control.

Results

Cloning and Protein Production:

Produced protein variants as schematically described in FIG. 4 where “Z”can be represented by SEQ ID NO:745 and SEQ ID NO:748-757 were analyzedby MALDI-TOF MS and on SDS-PAGE. (FIG. 5)

Inhibition of Hemolysis:

A subset of C5 binding Z variants were assayed for C5 binding activityin vitro and inhibition of hemolysis in sheep erythrocytes. Theconcentration of Z variant resulting in 50% inhibition of hemolysis(IC₅₀) or 50% inhibition of tracer binding to human C5 was calculated.Representative concentration-response curves for Z variants shown as SEQID NO:745 and SEQ ID NO:748-757 inhibiting hemolysis as described in themethods section are shown in FIGS. 6A and 6B. The result for different Zvariants fused to ABD094 (SEQ ID NO:759) via a short GS-linker are shownin FIG. 6A.

The parental Z variant Z05477a (SEQ ID NO:745) fused to ABD094 (SEQ IDNO:759) separated by a short GS linker exhibited an IC₅₀ value of about100 nM, whereas the tested second-generation C5 binding Z-ABD variantstypically inhibited hemolysis with IC₅₀ values around or below 1 nM.This suggests a more than 100-fold increase in potency for the C5binding Z variants identified in the maturation selection and subsequentscreening.

In FIG. 6B, C5 binding is shown for various combinations of onerepresentative Z variant (Z06175a; SEQ ID NO:753) alone, as a dimer andin fusion with ABD094 (SEQ ID NO:759) either in the N-terminus or theC-terminus via different linkers as specified in the figure. The C5binding combinations exhibited IC₅₀ values ranging from 86 pM to 12 nMwith human serum as measured using the above described assay. Thecorresponding value for the tic protein OmCI was typically 300 to 500pM.

In Vitro Kinetics:

Kinetic studies of binding characteristics for a number of Z variants(SEQ ID NO:748-757) optionally fused to ABD094 (SEQ ID NO:759), toimmobilized hC5, as well as to C5 in the presence of human albumin, wereperformed using the Biacore T200 instrument.

Data for ten different Z variants fused to ABD094 via a GS linker arepresented in Table 6.

TABLE 6 Human C5-binding characteristics for different Z-ABD fusions SEQID NO: Construct # of Z variant k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) Z-GS-SEQ ID NO: 748 6.93 × 10⁵ 9.04 × 10⁻⁴ 1.31 × 10⁻⁹ ABD094 SEQ ID NO: 7496.75 × 10⁵ 1.23 × 10⁻³ 1.83 × 10⁻⁹ SEQ ID NO: 750 7.65 × 10⁵ 1.34 × 10⁻³1.75 × 10⁻⁹ SEQ ID NO: 751 6.90 × 10⁵ 1.29 × 10⁻³ 1.87 × 10⁻⁹ SEQ ID NO:752 7.02 × 10⁵ 1.81 × 10⁻³ 2.58 × 10⁻⁹ SEQ ID NO: 753 7.90 × 10⁵ 1.01 ×10⁻³ 1.18 × 10⁻⁹ SEQ ID NO: 754 5.00 × 10⁵ 1.14 × 10⁻³ 2.28 × 10⁻⁹ SEQID NO: 755 6.84 × 10⁵ 2.08 × 10⁻³ 3.05 × 10⁻⁹ SEQ ID NO: 756 3.17 × 10⁵6.37 × 10⁻³ 2.01 × 10⁻⁹ SEQ ID NO:757 4.63 × 10⁵ 1.08 × 10⁻³ 2.34 × 10⁻⁹

Binding of the same Z variant (SEQ ID NO:753) but in differentconstructs; i.e. with/without ABD and different linkers, were alsoanalyzed using Biacore T200. In addition, the effect of albumin on someZ-ABD fusions was also assessed by running the same analysis in theabsence and in the presence of human albumin. These data are presentedbelow in Table 7.

TABLE 7 Human C5-binding characteristics for a Z-ABD fusion variant Z06175a (SEQ ID NO: 753, abbreviated Z) comprised in different constructs. Human Construct albumin k_(a) (1/Ms)k_(d) (1/s) K_(D) (M) Z-GS-ABD094 − 7.37 × 10⁵ 1.06 × 10⁻³ 1.43 × 10⁻⁹Z-GS-ABD094 + 6.74 × 10⁵ 9.62 × 10⁻⁴  1.43 × 10⁻⁹ Z-Z-GS-ABD094 − 5.93 ×10⁵ 3.74 × 10⁻⁴  6.30 × 10⁻¹⁰ Z-Z-GS-ABD094 + 6.02 × 10⁵ 4.67 × 10⁻⁴ 7.76 × 10⁻¹⁰ Z-GS-ABD094- − 8.69 × 10⁵ 5.75 × 10⁻⁴  6.62 × 10⁻¹⁰GSGGGGSGGGGS-Z (SEQ ID NO. 777) Z-GS-ABD094- + 6.55 × 10⁵ 3.83 × 10⁻⁴ 5.86 × 10⁻¹⁰ GSGGGGSGGGGS-Z (SEQ ID NO. 777) Z-Z-GSGGGGSGGGGS- − 4.59 ×10⁵ 6.32 × 10⁻⁴ 1.38 × 10⁻⁹ ABD094 (SEQ ID NO. 778) Z-Z-GSGGGGSGGGGS- +8.32 × 10⁵  9.39 × 10⁻⁴ 1.13 × 10⁻⁹ ABD094 (SEQ ID NO. 778) Z-GS −2.42 × 10⁶ 1.40 × 10⁻³ 5.79 × 10⁻¹⁰ Z-GSGGGGSGGGGS- − 3.64 × 10⁵ 1.37 ×10⁻³ 3.75 × 10⁻⁹ ABD094 (SEQ ID NO. 779)

Surprisingly small effects could be seen when comparing the affinitiesof the constructs for hC5 (SEQ ID NO:760) in the presence and absence ofalbumin. This suggests that simultaneous binding of albumin to the ABDmoiety of the constructs does not interfere with C5 interaction.

Steady State Binding of C5 Binding Z Variants to C5 Coated ECL Plates:

Steady state binding of C5 binding constructs composed of different Zvariants (SEQ ID NO:745 and 748-757), optionally fused to ABD094 (SEQ IDNO:759) in constructs as specified in FIG. 4, to hC5 was assessed in acompetition assay. By competing for binding to C5 coated on ECL plateswith a SULFO-TAG labeled C5 binding Z variants (SEQ ID NO:748) fused toABD (SEQ ID NO:759), steady state binding of the C5 constructs wasevaluated. As a comparison the tic protein OmCI (SEQ ID NO:761) was alsoincluded. The labeled Z-ABD variant containing SEQ ID NO:748 had anaffinity (K_(d)) of 0.9 nM for hC5. This labeled Z-ABD variant wasfurther found to bind to an antibody specific for the constant region ofZ variants in a concentration-dependent manner with a K_(d) of 0.34 nM.

The C5 binding Z-variants (SEQ ID NO:748-757) fused in the carboxyterminus to ABD094 (SEQ ID NO:759) by a GS linker were found to displace200 pM SULFO-TAG labeled Z-ABD variant with IC₅₀ values ranging fromabout 300 pM to 1 nM (FIG. 7A), whereas the corresponding constructcontaining the parental Z variant Z05477a (SEQ ID NO:745) exhibited anaffinity IC₅₀ value of about 30 nM. In contrast, the naturally occurringC5 binding protein OmCI was found to bind hC5 with an IC₅₀ of 1.5 nM(FIG. 7A). Thus, all the tested second-generation Z variants (SEQ IDNO:748-757) exhibited a higher binding affinity for human C5 than theparental Z variant Z05477a (SEQ ID NO:745). In addition, the affinitieswere higher than that of OmCI binding to human C5 using the same method.

A number of different constructs containing the same C5 binding domainas a monomer, dimer, with or without ABD as well as a few differentlinkers between the different domains were also tested (FIG. 7B).Monomeric variants of Z06175a (SEQ ID NO:753, optionally fused to aHis₆-tag or a C-terminal ABD) and the dimeric variants with a C-terminalABD linker were found to displace 200 pM SULFO-TAG labeled Z-ABD variantwith IC₅₀ values ranging from about 500 pM to 1.7 nM whereas the dimericvariant without an ABD and the monomeric variant with a N-terminal ABDdisplaced 200 pM SULFO-TAG labeled Z-ABD with IC₅₀ values of 4 nM and 17nM, respectively.

Selectivity:

Selectivity was addressed using SPR analysis and the surface with theimmobilized Z-ABD variant (SEQ ID NO:748 fused to SEQ ID NO:759 by aGS-linker) displayed no significant SPR signal when subjected to 40 and400 nM of the C5 paralogs human C3 and C4 as well as human IgG. As acomparison, 400 nM human C5 elicited an SPR response of about 450 RUshowing that the tested Z-ABD variant indeed is selective for C5 overC3, C4 and IgG.

Example 7: Interaction Studies of Z-ABD Variants with HSA, BSA and SerumAlbum from Rat and Mouse

Materials and Methods

Two different methods, size exclusion chromatography and Biacore, wereused to study the interaction between the albumin binding domain ABD094fused to a C5 binding Z variants.

Size exclusion chromatography (SEC) was employed to study theinteraction between Z06175a-GS-ABD094 (SEQ ID NO:753 fused to SEQ IDNO:759 by a GS linker) and HSA. Briefly, equimolar amounts ofZ06175a-GS-ABD094 and recombinant HSA (Novozymes) were preincubated inPBS at room temperature for 60 minutes and subsequently run on aSuperdex200 column (GE Healthcare) using the SMART system (GEHealthcare). Z06175a-GS-ABD094 and HSA were also run separately ascontrols.

Binding to immobilized albumin was studied using a Biacore 2000instrument (GE Healthcare). Recombinant human albumin (RECOMBUMIN,Novozymes) was coupled to a CM5 sensor chip (385 RU) using aminecoupling chemistry as described by the manufacturer. The coupling wasperformed by injecting human albumin in 10 mM Na-acetate buffer pH 4.5(GE Healthcare). The reference cell was treated with the same reagentsbut without injecting human albumin Injection of HBS-EP was alsoincluded as control so that the sensorgrams were double blanked.Experiments were performed in HBS-EP buffer, 10 mM glycine-HCl pH 2 (GEHealthcare) was used for regeneration, the flow rate was 30 μl/min anddata were collected at 25° C. Two different constructs were tested,Z-ABD (Z06175a-GS-ABD094) and Z-ABD-Z(Z06175a-GS-ABD094-GSGGGGSGGGGS-Z06175a) (SEQ ID NO. 780) at threedifferent concentrations; 25 nM, 100 nM and 400 nM. BIAevaluationversion 4.1.1 was used for evaluation of sensorgram data. In a similarfashion, binding of Z-ABD (Z06175a-GS-ABD094) to surfaces immobilizedwith serum albumin from rat (A4538, Sigma), mouse (A3559, Sigma), andcow (BSA, Sigma) was also investigated.

Results

On a SEC column, larger molecules elute faster than small. As seen inFIG. 8A, the co-injected HSA+ Z06175a-GS-ABD094 elute faster than whenHSA is injected alone suggesting that the two molecules behave as astable complex under these conditions. The smaller Z06175a-GS-ABD094elute slower than either the complex or HSA alone showing that theseproteins alone are smaller than the complex.

Biacore 2000 data for the analyzed Z-ABD and Z-ABD-Z variants show thatthe Z-ABD has a faster on-rate than when ABD is flanked by Z-domains oneither side (FIG. 8B). Analysis of the binding affinity of ABD fused Zdomains points at an affinity below 1 nM for Z-ABD whereas the Z-ABD-Zvariant bind to immobilized HSA with a K_(D) above 1 nM.

Z06175a-GS-ABD094 bound to rat serum albumin with very high affinity(KD<100 pM) whereas the interaction with immobilized mouse serum albuminwas weaker (KD of about 4 nM) than both with human and rat serum albuminInteraction with bovine serum albumin was not measurable.

These data agree well with published data on an earlier variant of ABD(Jonsson et al. Protein Engineering, Design & Selection 2008, 21:515-527) and show that the tested Z-ABD variant is strongly bound toserum albumin in human at clinically relevant concentrations as well asin mouse and rat allowing comparisons of pharmacokinetic data betweenanimals and humans.

Example 8: Pharmacokinetic Studies of C5 Binding Z Variant in Rats

Materials and Methods

Rodent in-Life Phase:

The pharmacokinetics of two C5 binding constructs Z-ABD(Z06175a-GS-ABD094; SEQ ID NO:753 fused to SEQ ID NO:759 by a GS linker,FIG. 4, construct 2) and Z-ABD-Z (Z06175a-GS-ABD094-GSGGGGSGGGGS-Z06175a(SEQ ID NO. 781); (SEQ ID NO:753 fused to SEQ ID NO:759 by a GS linker,followed by a GS(G₄S)₂ linker and a second SEQ ID NO:753 motif, FIG. 4,construct 5) was studied in Male Sprague Dawley (SD) rats (250-300 gbody weight). Each rat was given a single dose administration, i.v. (250nmol/kg) or s.c. (500 nmol/kg), of Z-ABD or A-ABD-Z (n=3 per dosegroup). Blood samples (200 μL) were drawn at 5, 20, and 45 min, as wellas 1.5, 4, 7, 24, 48, 72, 120, 168, 240, and 336 h followingadministration for the i.v. group and at 15 and 30 min, 1, 2, 4, 7, 24,48, 72, 120, 168, 240, and 336 h following administration for the s.c.group. Blood was collected in tubes and placed in the fridge for 20 minto allow clotting. Serum was subsequently harvested followingcentrifugation at 4000 rpm for 10 minutes. Serum samples were kept at−70° C. pending analysis.

Determination of C5 Binding Z Variant Concentrations in Serum Samplesfrom Animals Using LC/LC/MS/MS:

Serum concentrations of the administrated C5 binding constructs Z-ABDand Z-ABD-Z, as described above, were determined by mass spectrometry(LC/LC/MS/MS). Serum or plasma samples (25 μl) were diluted with 150 μlof a pepsin agarose (7 mg/ml, Sigma, cat. no. P0609) suspended in 1 Mammonium formate buffer pH 3.0 in a 500 μl Eppendorf tube. The tubeswere capped and agitated in an Eppendorf thermomixer compact at 37° C.for 20 min Following agitation, 25 μl of an internal standard solutionI(¹³C₆;¹⁵N)NKLDDDPSQSSEL (SEQ ID NO. 782) (amino acids 31-44 of the SEQID NO:746-757) (Thermo Fisher Scientific GmbH), diluted to 0.5 μM in0.1% trifluoroacetic acid (TFA), was added. Following addition ofinternal standard, the samples were mixed and filtered through 0.45 μmcellulose spin filters (Grace).

Standard samples for calibration were prepared by weighing 20 μl ofprotein stock solution with known protein concentration (5-10 mg/ml)followed by dilution with blank plasma from the species to be analyzed.The first stock plasma standard (3 μM) was diluted further down to 0.1μM.

40 μl of the samples were injected into a coupled column system followedby tandem mass spectrometry with multiple reaction monitoring (MRM). Thefirst column was an Ascentis RP-Amide column packed with 5 μm particles(2.1×150 mm, Supelco). An enrichment column; a Brownlee newgard column(3.2×15 mm) packed with 7 μm C18 particles, was used to trap the analytepeptide fraction from the first column. The effluent from the firstcolumn was diluted with 1 ml/min water pumped by Shimadzu pump into awhirl mixer (Lee Scientific). The last column was a mixed mode reversedphase and cation exchange column (2.1×100 mm) packed with 5 μm particlesPrimesep 100 (SIELC Inc).

The mobile phases for the first column (RP-Amide) provided on a firstliquid chromatograph (Acquity UPLC) were A: 2% acetonitrile, 0.1% aceticacid, 0.1% TFA, and 97.8% water, and B: acetonitrile with 0.1% aceticacid and 0.02% TFA. The flow was 0.5 ml/min and a linear gradient wasused for elution. The sample was eluted at isocratic conditions with100% A for 1 min, followed by 80% A at 7.9 min. At 8.1 min, the columnwas washed with 100% B for one minute, followed by reconditioning with100% A. The effluent from the column was connected to a Valco six portvalve controlled from the mass spectrometer software.

The trap column (3.2×15 mm) was connected to the six port valve in backflush mode. The mobile phases for the second column, provided on asecond liquid chromatograph (Agilent 1100), were A: 80% acetonitrile,19.9% water, and 0.1% formic acid, and B: 80% acetonitrile, 19% water,0.5% acetic acid and 0.5% TFA pumped by an Agilent 1100 liquidchromatograph at 0.5 ml/min and eluted with the following gradient: 100%A during the first 5 minutes followed by B gradually being raised from 0to 40% from 5 to 10 minutes followed by a raise to 100% B during thenext 6 seconds (10 to 10.1 minutes). B was kept at 100% until 11.5minutes followed by a drop to 0% (100% A) during the next 6 seconds(11.5 to 11.6 minute) and kept at 0% B throughout the cycle untilstopped at 13 minutes.

The effluent from the last column was connected to a triple quadrupolemass spectrometer (Sciex API 4000) equipped with an electrospray ionsource operated in positive ion mode. The MRM transitions were780.9>814.4 for the analyte and 784.5>821.4 for the internal standard.The declustering potential was optimized at 55 V and the collisionenergy to 35 V. The effective collision energy was 70 eV since theprecursor ion was doubly charged giving a singly charged fragment ion.The peak area ratios between the analyte and internal standard were usedfor quantification. Linear calibration curves were obtained with arecovery of 85% and a limit of quantification of about 40 nM.

Ex Vivo Hemolysis:

An ex vivo hemolytic assay for complement activation was performed inorder to optimally assemble in vivo conditions for the serum samplesfrom the above described in vivo studies. The serum samples were 5×diluted in a total reaction volume of 25 μl/well comprising 5 millionantibody sensitized sheep erythrocytes (EA). In general, a portion of 20μl EA suspension containing all other components (see Example 6) wasmixed (agitation 10 minutes) with 5 μl serum sample to initiate thehemolytic activation at 37° C. For mouse serum samples, such as inexample 11, however, 1 μl C5D had to be included in the 20 μl EAsuspension. The ex vivo assay was performed essentially as described forthe in vitro assay of Example 6. Calculations: Evaluation of thepharmacokinetic parameters was based on individual serum concentrationdata, the mean (±stdev) is reported for each dose group. Levels belowlower limit of quantitation (LLOQ) appearing at terminal sampling pointswere omitted from the pharmacokinetic analysis. Maximum serumconcentration, C_(max), and time to observed maximum serumconcentration, t_(max), were obtained directly from the serumconcentration data. The pharmacokinetic parameters; area under curve(AUC, AUC_(o-∞) and AUC_(0-last) calculated by the linear trapezoidalmethod), subcutaneous bioavailability (F, calculated as(AUC_(sc)/AUC_(iv))*(Dose_(iv)/Dose_(sc))), terminal serum half-life(T_(1/2,z), calculated as ln 2/λ_(z) where estimation of terminal slope,λ_(z), was based on at least 4 C=f(t) observations), mean residence time(MRT, calculated as AUMC/AUC), serum clearance (CL, calculated asDose/AUC_(0-∞)), volume of distribution at steady state (V_(ss),calculated as CL*MRT) and volume of distribution at the terminal phase(V_(z), calculated as CL/2) were calculated using WinNonlin softwareversion 5.2.1 (Pharsight Corp., USA), Non-Compartmental-Analysis.

Results

The pharmacokinetic data for Z-ABD and Z-ABD-Z following i.v. (250nmol/kg) and s.c. (500 nmol/kg) administration are summarized in Table8. Z-ABD was quantifiable in serum up to 10-14 days post dose in thei.v. group and 14 days in the s.c. group whereas Z-ABD-Z wasquantifiable in serum up to 10 days post dose in both dose groups (FIG.9). 14 days was the final sampling time point.

Correlating the serum concentration of C5 binding polypeptide with theamount of hemolysis in sheep erythrocytes, it was found that fullinhibition of hemolysis under the conditions described (e.g. serumdilution 1:5) was obtained by Z-ABD at serum concentrations above 1 μM(FIG. 12) whereas Z-ABD-Z reached full inhibition at serumconcentrations around 0.5 μM (FIG. 13). Surprisingly, as seen in FIG. 9and Table 8, Z-ABD has a lower serum clearance, a longer terminal serumhalf-life and a higher bioavailability than Z-ABD-Z. In terms of timethis lead to full inhibition of hemolysis for about three days afteradministration of 250 nmol/kg Z-ABD (FIG. 10) i.v. or 500 nmol/kg s.c(FIG. 11) to S.D.rats.

TABLE 8 Mean (±stdev) pharmacokinetics of Z-ABD and Z-ABD-Z followingi.v. and s.c. administration in male Sprague Dawley rats. Z-ABD Z-ABD-ZAdministration route i.v. s.c. i.v. s.c. Dose nmol/kg 250 500 250 500C_(max) μM 2.8 (0.2) 0.90 (0.10) T_(max) h 18 (9.8) 17 (12) AUC_(0-∞)μM*h 233 (34) 252 (11) 79 (7.5) 64 (1.2) AUC_(0-last) μM*h 226 (37) 247(11) 79 (6.9) 63 (1.0) F % 55 (3.1) 41 (2.6) T_(1/2, z) h 58 (4.6) 57(4.2) 36 (0.6) 46 (1.2) MRT h 69 (2.6) 80 (4.6) 27 (1.5) 63 (2.6) CLmL/h*kg 1.1 (0.2) 3.2 (0.2) V_(ss) mL/kg 73 (12) 83 (10) V_(z) mL/kg 90(18) 159 (12)

Example 9: Pharmacokinetic Studies of C5 Binding Z Variants in Monkey

Materials and Methods

The study in life phase was performed at Charles River, Nevada(www.criver.com), formulation of administered drug and analysis ofserums samples were performed in house. The pharmacokinetics of a Z-ABDvariant (Z06175a (SEQ ID NO:753) fused to ABD094 (SEQ ID NO:759) by a GSlinker) was investigated in the male Cynomolgus monkey (n=3) followingi.v. (intravenous) and s.c. (subcutaneous) administration. Evaluation ofthe pharmacokinetic parameters was performed according to Example 8,however following i.v. administration the initial serum half-life(T_(1/2α)) corresponding to the initial slope of the log-linear serumconcentration-time curve, intermediate serum half-life (T_(1/2β))corresponding to the slope of the log-linear serum concentration-timecurve associated with the secondary (intermediate) phase and terminalserum half-life (T_(1/2γ)) corresponding to the terminal slope of thelog-linear serum concentration-time curve was determined. T_(1/2) wascalculated as ln 2/λ where estimation of the slope, λ, was based on atleast 4 C=f(t) observations. The pharmacokinetic data presented for scadministration are compensated for pre-dose levels of Z-ABD while thegraph displaying serum concentration versus time after sc administrationshow the actual serum concentrations determined. The monkeys were 2-4years old with a body weight of 2.3-3 kg. Each monkey received a singlei.v. dose (540 nmol/kg) followed by a single s.c. dose (1635 nmol/kg)three weeks after the i.v. administration. Blood samples were taken at10 and 30 minutes and 1, 2, 4, 8, 24, 48, 72, 120, 168, 240, 336 and 504hours post dose following both administrations. The blood samples wereallowed to clot for 20-40 minutes in room temperature and thencentrifuged at 1500 to 2200 RCF at 2-8° C. for 10-15 minutes before theserum was harvested and frozen. The serum samples were stored at atemperature below −20° C. until analysis.

Serum concentrations of Z-ABD were analyzed by LC/LC/MS/MS as describedin Example 8. Serum concentrations determined by LC/LC/MS/MS were alsoconfirmed by a quantitative sandwich enzyme immunoassay technique. Apolyclonal antibody specific for the Z compartment of Z-ABD was coatedon to a microplate. Unbound polyclonal antibody was washed away andcasein was added as blocking agent to reduce unspecific binding to theplastic surface. Samples and standards were diluted in PBS containing0.5% casein and between 1-5% monkey normal serum. After washing awayunbound casein, standards and samples were pipetted to the wellsallowing any Z-ABD, presumed mainly to be associated with serum albumin,present in the sample to bind to the immobilized antibody. After washingaway any unbound Z-ABD, an HRP labeled polyclonal antibody specific foralbumin was added to detect the immobilized Z-ABD-albumin complex bycolorometric methods. Unbound polyclonal antibody was washed away and asubstrate solution was added to the wells and color develops inproportion to the amount of Z-ABD bound. Evaluation and calculation ofpharmacokinetic parameters were performed as described in Example 8.

Ex vivo hemolysis in serum from cynomolgus monkeys dosed with abovedescribed Z-ABD variant was monitored using the method described inExamples 6 and 8 with the modification that the monkey serum was dilutedonly two-fold compared to five-fold for rodent serum.

Results

Data on the mean (±stdev) pharmacokinetics of each dose group arepresented. Serum concentrations of Z-ABD were quantifiable at all timepoints following both i.v. and s.c. administration by LC/LC/MS/MS (FIG.14). ELISA data and LC/LC/MS/MS data correlated linearly by acoefficient of 0.986 but LC/LC/MS/MS data were used for thecalculations. Following i.v. administration of Z-ABD the initial serumhalf-life was 9.1 (0.8) hours, intermediate serum half-life was 84 (4)hours and the terminal serum half-life was 198 (51) hours. The meanresidence time was 246 (62) hours. The volume of distribution, V_(ss)and V_(z) was calculated to 110 (23) ml/kg and 127 (27) ml/kgrespectively and clearance was estimated to 0.45 (0.02)mL/h*kg.

Following s c administration, and corrected for pre-dose serum levelsremaining from the i v administration, maximum serum concentrations(mean C_(max) 21(3) μM) were reached at 8-24 h after dose. The terminalserum half-life was 206 (40) hours and the mean residence time was 250(68) hours. The subcutaneous bioavailability was estimated to be above70%.

The pharmacodynamic effect of the injected Z-ABD variant (Z06175a (SEQID NO:753) fused to ABD094 (SEQ ID NO:759) by a GS linker) was monitoredby hemolysis. The hemolytic effect in cynomolgus monkey was completelysuppressed (<20% of pre-dose) for at least seven days afteradministration of 5 mg/kg Z-ABD i.v. and 15 mg/kg Z-ABD s.c.

Example 10: In Vivo Studies Using Zymosan Induced Peritonitis

Materials and Methods

Administration to Mice:

C57BL/6 female mice received different concentrations of a Z-ABD fusionmolecule (Z06175a-GS-ABD094, SEQ ID NO:753 fused to SEQ ID NO:759 by aGS linker) or the positive control OmCI intraperitoneally (i.p.) 1 hourbefore induction with zymosan, or subcutaneously (s.c.) 18 hours beforeinduction with zymosan.

0.8 mg/mouse zymosan was administered i.p. 1 hour later orbital bloodsamples (in serum vials with coagulation activator) were taken underisoflurane anaesthesia. The animals were killed by cervical dislocation.A skin incision was made, and the abdominal muscular wall wasvisualized. PBS solution (including 2 mM EDTA) was gently injected intothe abdominal cavity. The abdomen was massaged and a sample of fluid(1-2 ml) was withdrawn. The samples were transferred to test tubes andstored on wet ice before centrifugation at 600 g for 10 min. Totalprotein and C5a concentrations in the supernatant were analyzed.

Blood samples were kept in a refrigerator for at least 30 min andcentrifugation was thereafter performed at 2000 g. Serum samples werestored in freezer (−70° C.) for later analysis of hemolytic activity andlevels of Z06175a-GS-ABD094.

Analysis of Hemolysis Activity in Serum Samples from Animals:

Analysis of hemolysis activity was performed according to the hemolysisassay described in Examples 6 and 7.

Analysis of C5a Concentration in Lavage from Mice Dosed with Zymosan andC5 Binding Z-ABD Fusion Molecules:

For detection of C5a in mouse peritoneal lavage samples, microtiterplates (MaxiSorp, Nunc) were coated overnight at 4° C. with 100 μl/wellof anti-05a antibody (cat. no. MAB21501, R&D Systems) at a concentrationof 1 μg/ml in 0.05 M sodium carbonate-bicarbonate buffer, pH 9.6 (cat.no. C-3041, Sigma). The plates were washed three times with PBScontaining 0.05% Tween 20 (PBST, cat. no. 09-9410-100, Medicago) andblocked with 200 μl/well of 1% BSA (cat. no. A7030, Sigma) in PBST for1-1.5 hat RT during agitation at 450 rpm. The plate was again washedthree times with PBST and then incubated with 100 μl/well of recombinantmouse C5a standard (cat. no. 2150-05, R&D Systems) at variousconcentrations in PBST with 0.1% BSA or samples for 2 h at RT duringagitation at 450 rpm. High concentration samples were also diluted inPBST with 0.1% BSA. The plate was once again washed three times withPBST and then incubated with 100 μl/well of biotinylated anti-C5aantibody (cat. no. BAF2150, R&D Systems) at a concentration of 0.1 μg/mlfor 1.5 h at RT while shaking the plate at 450 rpm. Following 3× washingwith PBST, the plate was incubated with 100 μl/well of streptavidin-HRP(cat. no. DY998, R&D Systems) at a 200 fold dilution in blocking bufferfor 20 min at RT during agitation at 450 rpm. After three final washes,the plate was developed with 100 μl/well TMB substrate (cat. no. T0440,Sigma) and read after 20-30 min at 650 nm using a Spectramax Plus platereader (Molecular Devices).

A standard curve was constructed by plotting the absorbance at 650 nmfor each standard against its concentration (range 0-4000 pg/ml).

Determination of Z Variant Concentration in Serum Samples from AnimalsUsing ECL:

Serum concentrations of administrated C5 binding Z06175a-GS-ABD094 (SEQID NO:753 fused to SEQ ID NO:759 by a GS linker) andZ06175a-GS-ABD094-GSGGGGSGGGS-Z06175a (SEQ ID NO: 789) (SEQ ID NO:753fused to SEQ ID NO:759 by a GS linker, followed by a GS(G₄S)₂ (SEQ IDNO: 785) linker and a second SEQ ID NO:753 motif, see FIG. 4 forconstruct description) were determined by ECL. Multi-array 96-wellhigh-bind, non-coated (Meso Scale Discovery cat. no. L15XB) plates werecoated with a goat anti-Affibody molecule Ig (Affibody AB, cat. no.20.1000.01.0005).

In similarity with Example 6, a Z-ABD variant (Z06009a, SEQ ID NO:748fused to ABD094, SEQ ID NO:759 Multi-array plates were coated with thegoat anti-Affibody molecule IgG (Affibody AB) overnight at 4° C., andsubsequently non-specific sites were blocked with PBS with 1% Casein fortwo hours at RT.

Meanwhile, serum samples were thawed from −70° C. and diluted in PBSwith casein in serum from the same animal strain. Standards and controlswere diluted in the corresponding buffer. Samples and standards wereincubated for three hours at RT while shaking the plate at 300 rpm.Incubation was terminated by washing 3×150 μL ice-cold PBS-Tween20.Immediately after the final wash, 150 μl 2× reading buffer (4× readingbuffer T, Meso Scale Discovery cat. no. R92TC-3 diluted 1:1 in ultrapureH₂O) was added to each well and the signal was detected using a platereader (SECTOR Imager 2400, Meso Scale Discovery).

In an alternative experiment, plates were coated with human C5 (SEQ IDNO:760, 1 pmol/well). Prior to addition to the coated plate, serumsamples and standards, diluted in serum or in serum and PBS with casein(all samples and standards were matched to the same serumconcentration), were heated to 60° C. for 30 min in order to denatureendogenous C5. This alternative experiment provided a method forexclusive detection of C5 binding proteins, whereas the antibodydependent strategy described above can be applied to all proteinsbinding to that particular antibody.

Results

Analysis of Serum Concentrations of Z-ABD and Hemolysis Activity inSerum Samples from Animals:

The serum concentrations as well as the ability to affect hemolysis insheep erythrocytes of the Z-ABD fusion molecule (Z06175a-GS-ABD094, SEQID NO:753 fused to SEQ ID NO:759 by a GS linker)) was assessed afteradministration of a low (20 nmol/kg), medium (100 nmol/kg) and high dose(500 nmol/kg). The serum concentrations were relatively linear withdose, and inhibition of hemolysis confirmed that the molecules in serumwere active and that the inhibition of hemolysis indeed also wasconcentration dependent.

Analysis of C5a Concentration in Lavage from Mice Dosed with Zymosan andC5 Binding Z-ABD Fusion Molecules:

The pro-inflammatory molecule zymosan was administered i.p. and in FIG.15 the effect on the highly inflammatory C5 cleavage product C5a inlavage as a function of zymosan dosing alone and zymosan dosed after adosing of a C5 binding Z variant at 20, 100 and 500 nmol/kg administereds.c. 18 h before zymosan treatment or OmCI administered i.p. 1 h beforezymosan treatment, is shown. Zymosan administration alone leads to apotent elevation of C5a in the lavage. This effect is blocked in a dosedependent manner by the presented C5 binding Z-ABD fusion molecule.

Example 11: Pharmacokinetic Studies of C5 Binding Protein in MiceFollowing Intratracheal Administration

Materials and Methods

The pharmacokinetic profile of the C5 binding constructZ06175a-GS-ABD094 (SEQID NO: 753 fused to SEQ ID NO:759 by a GS linker)following intratracheal administration to female C57b1 mice was studied.Temperature, relative humidity and lighting was set to maintain 22±1°C., 55±5% and a 12 h light—12 h dark cycle and diet and water wasprovided ad libitum. Animals were anesthetized with isoflurane and doseddirectly into the lungs using a microspray with 500 nmol/kgZ06175a-GS-ABD094. As much blood as possible was drawn, under anesthesiaby isoflurane, from vena cava at 5 min, 30 min, 1 h, 3 h, 7 h, 16 h, 24h, 48 h and 72 h (three animals/time point) for preparation of serumsamples. Serum samples were prepared by collecting blood in tubes andplacing the tubes in the fridge for 20 min. Subsequently, the tubes werecentrifuged at 4000 rpm for 10 minutes. A minimum of 100 μl serum wasprepared from each blood sample. Serum samples were kept at −70° C.prior to analysis. Serum concentrations of Z06175a-GS-ABD094 in eachsample was determined by ECL as described in Example 10 and the abilityof serum samples to affect hemolysis in sheep erythrocytes wasdetermined as described in Examples 6 and 8.

Results

The serum concentration in each sample and the corresponding ability toaffect hemolysis in sheep erythrocytes are described in FIG. 16A andFIG. 16B, respectively. Within 30 minutes, a plateau is reached withserum concentrations ranging from 300 to 1000 nM where hemolysis isnearly completely blocked. In serum sampled at time points later than 7h post-administration, hemolysis is gradually reoccurring. At the finaltime point three days after dosing, hemolysis was about 70% of control(FIG. 16B). These data clearly demonstrate absorption ofZ06175a-GS-ABD094 into the systemic circulation following intratrachealadministration and that the molecule functionally inhibits hemolysis.

Example 12: Pharmacokinetic Studies of C5 Binding Z Variant in RabbitEye Following Topical and Intravitreal Administration

Materials and Methods

Rabbit in-Life Phase:

The pharmacokinetics of a Z variant (Z06175a, SEQ ID NO:753 followed byGS (FIG. 4, construct 1)) was studied in rabbit eye followingintra-vitreal administration.

The study in-life phase and dissection of eyes from dosed animals(pigmented rabbits, 2-2.5 kg) was performed at Iris Pharma, La Gaude,France (www.iris-pharma.com). Animals were housed individually at 20±2°C. at 55±10% relative humidity with access to food and water ad lib.

Animals were divided in three groups: 1) intravitreal administration (50μl in each eye, n=3, six eyes totally) followed by dissection and serumsampling after one day, 2) intravitreal administration (50 μl in eacheye, n=3) followed by dissection and serum sampling after four days and3) untreated animals (n=5).

Four distinct eye compartments were dissected (aqueous humor, vitreous,neuro-retina and RPE-choroid) and immediately frozen at −80° C.Formulation of administered drug (20.2 mg/ml in 10 mM phosphate buffer,145 mM NaCl, pH 7.4) and analysis of drug in various eye compartmentswere performed in house.

Analysis of Z-Variant in Dissected Eye Compartments:

Dissected eye compartments were shipped on dry ice and stored at −80° C.until analysis. The retina and choroid samples were thawed in 10 times(volume/weight) PBS containing 1% human serum albumin in Lysing Matrix Dtubes (MP Biomedical) containing ceramic beads and agitated at speed 4for 2×20 s in a Savant Bio 101 homogenizer. The homogenate was removedfrom the beads using a pipette and transferred to a 1.5 ml Eppendorftube and centrifuged at 900 rpm for ten minutes. The aqueous humor andvitreous samples were treated the same way as retina and choroid withthe exception that no homogenization was needed. The vitreous samplesfrom groups one and two were diluted 10 times further in the same bufferas above. Five standards were prepared in PBS with HSA (35.8 μM, 3.58μM, 358 nM, 35.8 nM and 17.9 nM). Subsequently, standards and sampleswere subjected to pepsin digestion and analysis of the concentration ofZ variant in tissue extracts was determined using the LC/LC/MS/MS methoddescribed in Example 8.

Results

The concentrations of Z variant after intravitreal administration werehigh in all compartments after one day (6-200 μM) and, surprisingly,remained high 4 days post-administration (1.5-78 μM). In particular, theconcentration of the Z molecule in the vitreous ranged from 118 to 201μM (average 161 μM, n=6 eyes) one day after injection and remained at 26to 78 μM (average 46 μM, n=6) four days post-injection, pointing at aT_(1/2) of several days. There appears to be an inverse relationshipbetween size and elimination of drugs after intravitreal injection inrabbit eye described by the following examples; Moxifloxacin (MW<0.35kDa, T_(1/2)=1.72 h, Mohan et al. Trans Am Ophthalmol Soc 2005,103:76-83), ESBA105 (MW=26 kDa, T_(1/2)=25 h, Ottiger et al.Investigative Ophthalmology & Visual Science 2009, 50: 779-786) andRanibizumab (MW=48 kDa, T_(1/2)=2.88 days, Bakri et al. American Academyof Ophthalmology 2007, 114:2179-2182). The Z variant tested here had amolecular weight of 7.0 kDa, suggesting that the elimination of the Zmolecule was slower than what would be expected for such a smallmolecule in vitreous.

The invention claimed is:
 1. A C5 binding polypeptide, comprising a C5 binding motif, BM, which motif consists of an amino acid sequence selected from i) (SEQ ID NO: 763) EX₂X₃X₄A X₆X₇EID X₁₁LPNL X₁₆X₁₇X₁₈QW X₂₁AFIX₂₅ X₂₆LX₂₈D, 

wherein, independently of each other, X₂ is selected from H, Q, S, T and V; X₃ is selected from I, L, M and V; X₄ is selected from A, D, E, H, K, L, N, Q, R, S, T and Y; X₆ is selected from N and W; X₇ is selected from A, D, E, H, N, Q, R, S and T; X₁₁ is selected from A, E, G, H, K, L, Q, R, S, T and Y; X₁₆ is selected from N and T; X₁₇ is selected from I, L and V; X₁₈ is selected from A, D, E, H, K, N, Q, R, S and T; X₂₁ is selected from I, L and V; X₂₅ is selected from D, E, G, H, N, S and T; X₂₆ is selected from K and S; X₂₈ is selected from A, D, E, H, N, Q, S, T and Y; and ii) an amino acid sequence which has at least 86% identity to the sequence defined in i), wherein the polypeptide binds to C5.
 2. A C5 binding polypeptide according to claim 1, wherein the amino acid sequence i) fulfills at least four of the following eight conditions I-VIII: I. X₂ is V; II. X₃ is selected from I and L; III. X₆ is IV. X₇ is selected from D and N; V. X₁₇ is selected from I and L; VI. X₂₁ is L; VII. X₂₅ is N; VIII. X₂₈ is D.
 3. The C5 binding polypeptide according to claim 1, wherein the amino acid sequence is selected from any one of SEQ ID NO:1-248.
 4. The C5 binding polypeptide according to claim 3, wherein the amino acid sequence is selected from any one of SEQ ID NO:1-12, SEQ ID NO:20, SEQ ID NO:23-24, SEQ ID NO:26-28, SEQ ID NO:32-35, SEQ ID NO:38-39, SEQ ID NO:41, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:56-57, SEQ ID NO:59, SEQ ID NO:66, SEQ ID NO:78-79, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:106, SEQ ID NO:110, SEQ ID NO:119, SEQ ID NO:125, SEQ ID NO:141, SEQ ID NO:151, SEQ ID NO:161, SEQ ID NO:166, SEQ ID NO:187, SEQ ID NO:197, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:215 and SEQ ID NO:243.
 5. The C5 binding polypeptide according to claim 4, wherein the amino acid sequence is selected from any one of SEQ ID NO:1-12.
 6. The C5 binding polypeptide according to claim 1, in which said C5 binding motif forms part of a three-helix bundle protein domain.
 7. A C5 binding polypeptide, which comprises the amino acid sequence: (SEQ ID NO: 764) K-[BM]-DPSQS X_(a)X_(b)LLX_(c) EAKKL NDX_(d)Q; 

or an amino acid sequence which has at least 79% identity to SEQ ID NO: 764, wherein X_(a) is selected from A and S; X_(b) is selected from N and E; X_(c) is selected from A, S and C; X_(d) is selected from A and S; and wherein [BM] is a C5 binding motif, which motif consists of the amino acid sequence EX₂X₃X₄A X₆X₇EID X₁₁LPNL X₁₆X₁₇X₁₈QW X₂₁AFIX₂₅ X₂₆LX₂₈D (SEQ ID NO: 763), or an amino acid sequence which has at least 86% identity to SEQ ID NO:763, wherein independently of each other: X₂ is selected from H, Q, S, T and V; X₃ is selected from I, L, M and V; X₄ is selected from A, D, E, H, K, L, N, Q, R, S, T and Y; X₆ is selected from N and W; X₇ is selected from A, D, E, H, N, Q, R, S and T; X₁₁ is selected from A, E, G, H, K, L, Q, R, S, T and Y; X₁₆ is selected from N and T; X₁₇ is selected from I, L and V; X₁₈ is selected from A, D, E, H, K, N, Q, R, S and T; X₂₁ is selected from I, L and V; X₂₅ is selected from D, E, G, H, N, S and T; X₂₆ is selected from K and S; X₂₈ is selected from A, D, E, H, N, Q, S, T and Y; and wherein the [BM] motif binds to C5.
 8. The C5 binding polypeptide according to claim 7, wherein the amino acid sequence is selected from any one of SEQ ID NO:249-496.
 9. The C5 binding polypeptide according to claim 8, wherein the amino acid sequence is selected from any one of SEQ ID NO:249-260, SEQ ID NO:268, SEQ ID NO:271-272, SEQ ID NO:274-276, SEQ ID NO:280-283, SEQ ID NO:286-287, SEQ ID NO:289, SEQ ID NO:294, SEQ ID NO:297, SEQ ID NO:304-305, SEQ ID NO:307, SEQ ID NO:314, SEQ ID NO:326-327, SEQ ID NO:335, SEQ ID NO:340, SEQ ID NO:354, SEQ ID NO:358, SEQ ID NO:367, SEQ ID NO:373, SEQ ID NO:389, SEQ ID NO:399, SEQ ID NO:409, SEQ ID NO:414, SEQ ID NO:435, SEQ ID NO:445, SEQ ID NO:451, SEQ ID NO:453, SEQ ID NO:463 and SEQ ID NO:491.
 10. The C5 binding polypeptide according to claim 9, wherein the amino acid sequence is selected from any one of SEQ ID NO:249-260.
 11. The C5 binding polypeptide according to claim 10, wherein the amino acid sequence is selected from any one of SEQ ID NO:497-757.
 12. The C5 binding polypeptide according to claim 11, wherein the amino acid sequence is selected from any one of SEQ ID NO:497-508, SEQ ID NO:516, SEQ ID NO:519-520, SEQ ID NO:522-524, SEQ ID NO:528-531, SEQ ID NO:534-535, SEQ ID NO:537, SEQ ID NO:542, SEQ ID NO:545, SEQ ID NO:552-553, SEQ ID NO:555, SEQ ID NO:562, SEQ ID NO:574-575, SEQ ID NO:583, SEQ ID NO:588, SEQ ID NO:602, SEQ ID NO:606, SEQ ID NO:615, SEQ ID NO:621, SEQ ID NO:637, SEQ ID NO:647, SEQ ID NO:657, SEQ ID NO:662, SEQ ID NO:683, SEQ ID NO:693, SEQ ID NO:699, SEQ ID NO:701, SEQ ID NO:711, SEQ ID NO:739 and SEQ ID NO:746-757.
 13. The C5 binding polypeptide according to claim 12, wherein the amino acid sequence is selected from any one of SEQ ID NO:497-508 and SEQ ID NO:746-757.
 14. The C5 binding polypeptide according to claim 13, wherein the amino acid sequence is selected from any one of SEQ ID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:750 and SEQ ID NO:753.
 15. The C5 binding polypeptide according to claim 1, which inhibits cleavage of C5.
 16. The C5 binding polypeptide according to claim 1, wherein the C5 binding polypeptide binds to C5 such that the K_(D) value of the interaction is at most 1×10⁻⁶ M.
 17. The C5 binding polypeptide according to claim 1, comprising further C terminal and/or N terminal amino acids that improve production, purification, stabilization in vivo or in vitro, coupling, or detection of the polypeptide.
 18. The C5 binding polypeptide according to claim 1, wherein the polypeptide is in multimeric form, comprising at least two C5 binding polypeptide monomer units, the amino acid sequences of which may be the same or different.
 19. A C5 binding compound, comprising at least one C5 binding polypeptide according to claim 1; at least one albumin binding domain of streptococcal protein G, or a derivative thereof; and at least one linking moiety for linking said at least one albumin binding domain or derivative thereof to the C or N terminal of said at least one C5 binding polypeptide.
 20. The C5 binding compound according to claim 19, having a structure selected from [CBP1]-[L1]-[ALBD]; [CBP1]-[CBP2]-[L1]-[ALBD]; [CBP1]-[L1]-[ALBD]-[L2]-[CBP2]; [ALBD]-[L1]-[CBP1]; [ALBD]-[L1]-[CBP1]-[CBP2]; [CBP1]-[L1]-[CBP2]-[L2]-[ALBD]; and [ALBD]-[L1]-[CBP1]-[L2]-[CBP2] wherein, independently of each other, [CBP1] and [CBP2] are C5 binding polypeptides which may be the same or different; [L1] and [L2] are linking moieties which may be the same or different; and [ALBD] is an albumin binding domain of streptococcal protein G, or derivative thereof.
 21. The C5 binding compound according to claim 20, wherein the linking moiety is selected from G; GS; [G₂S]_(n); [G₃S]_(n) (SEQ ID NO:783); [G₄S]_(n) (SEQ ID NO:784); GS[G₄S]_(n) (SEQ ID NO:785); [S₂G]_(m); [S₃G]_(m) (SEQ ID NO:786); [S₄G]_(m) (SEQ ID NO:787); and VDGS (SEQ ID NO:788); wherein n is 0-7; and wherein m is 0-7.
 22. The C5 binding compound according to claim 19, wherein said albumin binding domain is as set out in SEQ ID NO:759.
 23. The C5 binding compound according to claim 19, wherein each of said C5 binding polypeptides is independently selected from any one of SEQ ID NO:497-757.
 24. A polynucleotide encoding a polypeptide according to claim
 1. 25. A polynucleotide encoding a compound according to claim
 19. 26. A combination of a C5 binding polypeptide according to claim 1 with a therapeutic agent.
 27. A combination of a C5 binding compound according to claim 19 with a therapeutic agent.
 28. A method of treatment of a C5 related condition, comprising administering a C5 binding polypeptide according to claim 1, a C5 binding compound according to claim 19, or the combination according to claim 26, to a mammalian subject in need thereof.
 29. The method of treatment according to claim 28, in which binding of the C5 binding polypeptide, the C5 binding compound or the combination to C5 inhibits cleavage of C5.
 30. The method of treatment according to claim 28, wherein said C5 related condition is selected from inflammatory disease; autoimmune disease; infectious disease; cardiovascular disease; neurodegenerative disorders; cancer; graft injury; wounds; eye disease; kidney disease; pulmonary diseases; hematological diseases; allergic diseases and dermatological diseases.
 31. The method of treatment according to claim 30, wherein said C5 related condition is paroxysmal nocturnal hemoglobinuria (PNH).
 32. The method of treatment according to claim 28, wherein said C5 binding polypeptide is administered intravenously, subcutaneously, by inhalation, nasally, orally, intravitreally, or topically.
 33. The C5 binding polypeptide according to claim 1, wherein the C5 binding polypeptide binds to C5 such that the K_(D) value of the interaction is at most 1×10⁻⁷M.
 34. The C5 binding polypeptide according to claim 1, wherein the C5 binding polypeptide binds to C5 such that the K_(D) value of the interaction is at most 1×10⁻⁸ M.
 35. The C5 binding polypeptide according to claim 1, wherein the C5 binding polypeptide binds to C5 such that the K_(D) value of the interaction is at most 1×10⁻⁹ M. 